Methods for treatment of metabolic disorders using epimetabolic shifters, multidimensional intracellular molecules, or environmental influencers

ABSTRACT

Methods and formulations for treating metabolic disorders in humans using epimetabolic shifters, multidimensional intracellular molecules or environmental influencers are described.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/177,241, filed May 11, 2009, entitled “Methods for Treatment ofOncological Disorders Using an Epimetabolic Shifter (Coenzyme Q10)”(Attorney Docket No.: 117732-00601), U.S. Provisional Application No.61/177,243, filed May 11, 2009, entitled “Methods for Treatment ofOncological Disorders Using Epimetabolic Shifters, MultidimensionalIntracellular Molecules or Environmental Influencers” (Attorney DocketNo.: 117732-00701), U.S. Provisional Application No. 61/177,244, filedMay 11, 2009, entitled “Methods for the Diagnosis of OncologicalDisorders Using Epimetabolic Shifters, Multidimensional IntracellularMolecules or Environmental Influencers” (Attorney Docket No.:117732-00801), U.S. Provisional Application No. 61/177,245, filed May11, 2009, entitled “Methods for Treatment of Metabolic Disorders UsingEpimetabolic Shifters, Multidimensional Intracellular Molecules orEnvironmental Influencers” (Attorney Docket No.: 117732-00901), and U.S.Provisional Application No. 61/177,246, filed May 11, 2009, entitled“Methods for the Diagnosis of Metabolic Disorders Using EpimetabolicShifters, Multidimensional Intracellular Molecules or EnvironmentalInfluencers” (Attorney Docket No.: 117732-01001), the entire contents ofeach of the aforementioned applications are hereby incorporated hereinby reference.

BACKGROUND OF THE INVENTION

The invention relates to the treatment, prevention, and reduction ofmetabolic disorders, such as diabetes and obesity.

As the levels of blood glucose rise postprandially, insulin is secretedand stimulates cells of the peripheral tissues (skeletal muscles andfat) to actively take up glucose from the blood as a source of energy.Loss of glucose homeostasis as a result of dysregulated insulinsecretion or action typically results in metabolic disorders such asdiabetes, which may be co-triggered or further exacerbated by obesity.Because these conditions are often fatal, strategies to restore adequateglucose clearance from the bloodstream are required.

Although diabetes may arise secondary to any condition that causesextensive damage to the pancreas (e.g., pancreatitis, tumors,administration of certain drugs such as corticosteroids or pentamidine,iron overload (e.g., hemochromatosis), acquired or geneticendocrinopathies, and surgical excision), the most common forms ofdiabetes typically arise from primary disorders of the insulin signalingsystem. There are two major types of diabetes, namely type 1 diabetes(also known as insulin dependent diabetes (IDDM)) and type 2 diabetes(also known as insulin independent or non-insulin dependent diabetes(NIDDM)), which share common long-term complications in spite of theirdifferent pathogenic mechanisms.

Type 1 diabetes, which accounts for approximately 10% of all cases ofprimary diabetes, is an organ-specific autoimmune disease characterizedby the extensive destruction of the insulin-producing beta cells of thepancreas. The consequent reduction in insulin production inevitablyleads to the deregulation of glucose metabolism. While theadministration of insulin provides significant benefits to patientssuffering from this condition, the short serum half-life of insulin is amajor impediment to the maintenance of normoglycemia. An alternativetreatment is islet transplantation, but this strategy has beenassociated with limited success.

Type 2 diabetes, which affects a larger proportion of the population, ischaracterized by a deregulation in the secretion of insulin and/or adecreased response of peripheral tissues to insulin, i.e., insulinresistance. While the pathogenesis of type 2 diabetes remains unclear,epidemiologic studies suggest that this form of diabetes results from acollection of multiple genetic defects or polymorphisms, eachcontributing its own predisposing risks and modified by environmentalfactors, including excess weight, diet, inactivity, drugs, and excessalcohol consumption. Although various therapeutic treatments areavailable for the management of type 2 diabetes, they are associatedwith various debilitating side effects. Accordingly, patients diagnosedwith or at risk of having type 2 diabetes are often advised to adopt ahealthier lifestyle, including loss of weight, change in diet, exercise,and moderate alcohol intake. Such lifestyle changes, however, are notsufficient to reverse the vascular and organ damages caused by diabetes.

Coenzyme Q10, also referred to herein as CoQ10, Q10, ubiquinone, orubidecarenone, is a popular nutritional supplement and can be found incapsule form in nutritional stores, health food stores, pharmacies, andthe like, as a vitamin-like supplement to help protect the immune systemthrough the antioxidant properties of ubiquinol, the reduced form ofCoQ10. CoQ10 is art-recognized and further described in InternationalPublication No. WO 2005/069916, the entire disclosure of which isincorporated by reference herein.

CoQ10 is found throughout most tissues of the human body and the tissuesof other mammals. The tissue distribution and redox state of CoQ10 inhumans has been reviewed in a review article by Bhagavan and Chopra(2006 Free Radical Research 40(5):445-453). The authors report that “asa general rule, tissues with high-energy requirements or metabolicactivity such as the heart, kidney, liver and muscle contain relativelyhigh concentrations of CoQ10.” The authors further report that “[a]major portion of CoQ10 in tissues is in the reduced form as thehydroquinone or uniquinol, with the exception of brain and lungs,” which“appears to be a reflection of increased oxidative stress in these twotissues.” In particular, Bhagavan report that in heart, kidney, liver,muscle, intenstine and blood (plasma), about 61%, 75%, 95%, 65%, 95% and96%, respectively, of CoQ10 is in the reduced form. Similarly,Ruiz-Jiminez et al. (2007 J. Chroma A, 1175, 242-248) report that whenhuman plasma was evaluated for Q10 and the reduced form of Q10 (Q10H2),the majority (90%) of the molecule was found in the reduced form.

CoQ10 is very lipophilic and, for the most part, insoluble in water.CoQ10 is very lipophilic and, for the most part, insoluble in water. Dueto its insolubility in water, limited solubility in lipids, andrelatively large molecular weight, the efficiency of absorption oforally administered CoQ10 is poor. Bhagavan and Chopra report that “inone study with rats it was reported that only about 2-3% oforally-administered CoQ10 was absorbed.” Bhagavan and Chopra furtherreport that “[d]ata from rat studies indicate that CoQ10 is reduced toubiquinol either during or following absorption in the intestine.”

Given that the strategies currently available for the management ofdiabetes are suboptimal, there is a compelling need for treatments thatare more effective and are not associated with such debilitatingside-effects.

SUMMARY OF THE INVENTION

The present invention is partly based on the finding that mitochondrialdysfunction is associated with a wide range of diseases, includingmetabolic diseases (such as diabetes and obesity), and that certainendogenous molecules, such as CoQ10, hold the key to the successfuldiagnosis, treatment, and prevention of such metabolic diseases. Theinvention is also partly based on the finding that these key endogenousmolecules play important roles in maintaining normal mitochondrialfunction by directly influencing oxidative phosphorylation, and thatrestoring or promoting more normalized mitochondrial osidativephosphorylation can effectively treat or prevent the progression ofmetabolic diseases. The invention is further based on the discovery thata class of environmental enfluencers (e.g., CoQ10) can selectivelyelicit, in disease cells of the metabolic diseases, a cellular metabolicenergy shift towards more normalized mitochondrial oxidativephosphorylation. These environmental influencers are capable ofmodulating intracellular targets that serve as key indices of metabolicdisorders (such as diabetes), in a manner representative of therapeuticendpoints.

The present invention is further based, at least in part, on thediscovery that application of endogenous Coenzyme Q10 (also referred toas CoQ10 or Q10 herein) to cells results in an apoptotic response. Theapoptotic response is preferentially induced in cancer cells. A time anddose response of mitochondrial Q10 levels was observed, wherein after 48hours, the level of Q10 in cell mitochondria was increased by six fold.The invention is further based on the surprising and unexpecteddiscovery that the Q10 is maintained in the supplied oxidized form(pro-oxidant) and not converted to the reduced (anti-oxidant) form ofQ10H2 in any significant amounts. The invention is based on the furtherdiscovery that a significant number of proteins and mRNA levels aremodulated in cells treated with Q10. These modulated proteins were foundto be clustered into several cellular pathways, including apoptosis,cancer biology and cell growth, glycolysis and metabolism, moleculartransport, and cellular signaling.

Applicants' data described herein has provided insight into themechanism of action of Q10. In particular, while not wishing to be boundby theory, Applicants' discoveries indicate that Q10 induces a metabolicshift to the cell microenvironment. Many diseases are known to beassocieated with an altered metabolic state. For example, differentialmetabolism is known to occur in cancer cells (the Warurg effect),whereby most cancer cells predominantly produce energy by glycolysisfollowed by lactic acid fermentation in the cytosol, rathe than byoxidative phosphorylation (oxidation of pyruvate) in the mitochondria.In another example, metabolic disorders, such as diabetes and obesity,are associated with an altered glucose metabolism.

Accordingly, the invention provides, in a first aspect, a method fortreating, alleviating symptoms of, inhibiting progression of, orpreventing a CoQ10 responsive disorder in a mammal, the methodcomprising: administering to the mammal in need thereof atherapeutically effective amount of pharmaceutical compositioncomprising at least one environmental influencer (env-influencer),wherein the environmental influencer selectively elicits, in a diseasecell of the mammal, a cellular metabolic energy shift towards levels ofglycolysis and mitochondrial oxidative phosphorylation observed in anormal cell of the mammal under normal physiological conditions.

In one embodiment, the CoQ10 responsive disorder is a metabolicdisorder.

The invention provides, in another aspect, a method for treating,alleviating symptoms of, inhibiting progression of, or preventing ametabolic disorder in a mammal, the method comprising administering tothe mammal in need thereof a therapeutically effective amount of apharmaceutical composition comprising at least one environmentalinfluencer (env-influencer), wherein the environmental influencerselectively elicits, in a disease cell of the mammal, a cellularmetabolic energy shift towards normalized mitochondrial oxidativephosphorylation.

In one embodiment, the environmental influencer does not substantiallyelicit, in normal cells of the mammal, the cellular metabolic energyshift towards mitochondrial oxidative phosphorylation.

In one embodiment, the mammal is human (or a non-human mammal).

In one embodiment, the metabolic disorder is responsive or sensitive totreatment by Coenzyme Q10 or its metabolites or analogs thereof.

In one embodiment, the metabolic disorder is characterized by adysregulated mitochondrial oxidative phosphorylation function that leadsto altered gene regulation and/or protein-protein interactions whichcontribute to or causally lead to the metabolic disease.

In one embodiment, the environmental influencer comprises (a)benzoquinone or at least one molecule that facilitates the biosynthesisof the benzoquinone ring, and (b) at least one molecule that facilitatesthe synthesis of and/or attachment of isoprenoid units to thebenzoquinone ring.

In one embodiment, said at least one molecule that facilitates thebiosynthesis of the benzoquinone ring comprises: L-Phenylalanine,DL-Phenylalanine, D-Phenylalanine, L-Tyrosine, DL-Tyrosine, D-Tyrosine,4-hydroxy-phenylpyruvate, 3-methoxy-4-hydroxymandelate(vanillylmandelate or VMA), vanillic acid, pyridoxine, or panthenol.

In one embodiment, said at least one molecule that facilitates thesynthesis of and/or attachment of isoprenoid units to the benzoquinonering comprises: phenylacetate, 4-hydroxy-benzoate, mevalonic acid,acetylglycine, acetyl-CoA, or farnesyl.

In one embodiment, the environmental influencer comprises (a) one ormore of L-Phenylalanine, L-Tyrosine, and 4-hydroxyphenylpyruvate; and(b) one or more of 4-hydroxy benzoate, phenylacetate, and benzoquinone.

In one embodiment, the environmental influencer: (a) inhibits Bcl-2expression and/or promotes Caspase-3 expression; and/or (b) inhibitscell proliferation.

In one embodiment, the environmental influencer is a multidimensionalintracellular molecule (MIM). In one embodiment, the MIM is selectedfrom: alpha ketoglutarate/alpha ketoglutaric acid, Malate/Malic acid,Succinate/Succinic acid, Glucosamine, Adenosine, Adenosine Diphosphate,Glucuronide/Glucuronic acid, Nicotinic Acid, Nicotinic AcidDinucleotide, Alanine/Phenylalanine, Pyridoxine, Thiamine, or FlavinAdenine Dinucleotide. In one embodiment, the multidimensionalintracellular molecule is selected from the group consisting of acetylCo-A, palmityl Co-A, L-carnitine, and amino acids, e.g., tyrosine,phenylalanine, and cysteine.

In one embodiment, the environmental influencer is an epimetabolicshifter (epi-shifter). In one embodiment, the epimetabolic shifter isselected from Transaldolase, Transketolase, Succinyl CoA synthase,Pyruvate Carboxylase, or Riboflavin. In one embodiment, the epimetabolicshifter is selected from the group consisting of coenzyme Q10, vitaminD3 and extracellular matrix components. In one embodiment, theepimetabolic shifter is coenzyme Q10. In one embodiment, theextracellular matrix components are selected from the group consistingof fibronectin, immunomodulators (e.g., TNFα or an interleukin),angiogenic factors, and apoptotic factors.

In one embodiment, a population of humans are treated and at least 25%of the population had a systemic environmental influencer level that wastherapeutic for the disorder being treated. In other embodiments, apopulation of humans are treated and at least 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% ormore of the population had a systemic Coenzyme Q10 level that wastherapeutic for the disorder being treated. It should be understood thatranges having any one of these values as the upper or lower limits arealso intended to be part of this invention, e.g., 10% to 25%, 15% to35%, 25% to 50%, 35% to 60%, 40% to 70%, 50% to 75%, 60% to 85% or 70%to 90%.

In one embodiment, the metabolic disorder being treated is not adisorder typically treated via topical administration with theexpectation of systemic delivery of an active agent at therapeuticallyeffective levels.

In one embodiment, the concentration of the environmental influencer inthe tissues of the human being treated is different than that of acontrol standard of human tissue representative of a healthy or normalstate.

In one embodiment, the form of the environmental influencer administeredto the human is different than the predominant form found in systemiccirculation in the human. In one embodiment, the environmentalinfluencer is administered to the human in oxidized form.

In one embodiment, the amount sufficient to treat the metabolic disorderin the human up-regulates or down-regulates mitochondrial oxidativephosphorylation.

In one embodiment, the amount sufficient to treat the metabolic disorderin the human modulates anaerobic use of glucose and/or lactatebiosynthesis.

The invention provides, in another aspect, a method for treating orpreventing a metabolic disorder in a human, comprising administering anenvironmental influencer to the human in an amount sufficient to treator prevent the metabolic disorder, wherein the environmental influenceris administered such that it is maintained in its oxidized form duringtreatment, thereby treating or preventing the metabolic disorder.

In one embodiment, the form of the environmental influencer administeredto the human is different than the predominant form found in systemiccirculation in the human.

The invention provides, in still another aspect, a method for treatingor preventing a metabolic disorder in a human comprising selecting ahuman subject suffering from a metabolic disorder; and administering tosaid human a therapeutically effective amount of an env-influencercapabable of augmenting mitochondrial oxidative phosphorylation and,optionally, blocking anaerobic use of glucose, thereby treating orpreventing the metabolic disorder.

The invention provides, in another aspect, a method for selectivelyaugmenting mitochondrial oxidative phosphorylation, in a disease cell ofa mammal in need of treatment for a metabolic disorder, the methodcomprising: administering to said mammal a therapeutically effectiveamount of a pharmaceutical composition comprising at least oneenv-influencer, thereby selectively augmenting mitochondrial oxidativephosphorylation in said disease cell of the mammal.

In one embodiment of the methods of the invention, the method furthercomprises upregulating the expression of one or more genes selected fromthe group consisting of the molecules listed in Tables 2-4 & Tables 6-28& Tables 63-68 with a positive fold change; and/or downregulating theexpression of one or more genes selected from the group consisting ofthe molecules listed in Tables 2-4 & Tables 6-28 & Tables 63-68 with anegative fold change, thereby treating or preventing the metabolicdisorder. In one embodiment, the method further comprises modulating theexpression of one or more genes selected from the group consisting ofHNF4-alpha, Bcl-x1, Bcl-xS, BNIP-2, Bcl-2, Birc6, Bcl-2-L11, XIAP, BRAF,Bax, c-Jun, Bmf, PUMA, cMyc, transaldolase 1, COQ1, COQ3, COQ6,prenyltransferase, 4-hydrobenzoate, neutrophil cytosolic factor 2,nitric oxide synthase 2A, superoxide dismutase 2, VDAC, Bax channel,ANT, Cytochrome c, complex 1, complex II, complex III, complex IV, Foxo3a, DJ-1, IDH-1, Cpt1C and Cam Kinase II.

In one embodiment of the methods of the invention, the treatment occursvia an interaction of the environmental influencer with a moleculeselected from the group consisting of the molecules listed in Tables 2-4& 6-28 & 63-68. In one embodiment, the treatment occurs via aninteraction of the environmental influencer with a protein selected fromthe group consisting of HNF4-alpha, Bcl-xl, Bcl-xS, BNIP-2, Bcl-2,Birc6, Bcl-2-L11 (Bim), XIAP, BRAF, Bax, c-Jun, Bmf, PUMA, cMyc,transaldolase 1, COQ1, COQ3, COQ6, prenyltransferase, 4-hydrobenzoate,neutrophil cytosolic factor 2, nitric oxide synthase 2A, superoxidedismutase 2, VDAC, Bax channel, ANT, Cytochrome c, complex 1, complexII, complex III, complex IV, Foxo 3a, DJ-1, IDH-1, Cpt1C and Cam KinaseII. In one embodiment, the treatment occurs via an interaction of theenv-influencer with HNF4alpha. In one embodiment, the treatment occursvia an interaction of the env-influencer with transaldolase.

In one embodiment of the methods of the invention, the metabolicdisorder is selected from the group consisting of diabetes, obesity,pre-diabetes, Metabolic Syndrome and any key elements of a metabolicdisorder.

In one embodiment, the metabolic disorder is diabetes, and theenv-influencer affects beta cell function, insulin metabolism, and/orglucagon deposition.

In one embodiment, the metabolic disorder is obesity, and theenv-influencer affects beta cell oxidation in the mitochondria, decreasein adipocyte size, and/or control of cortisol levels.

In one embodiment, the metabolic disorder is a cardiovascular disease,and the env-influencer affects decrease in smooth muscle cellproliferation in the tunica media, lipid peroxidation, thromboxane-ax2synthesis, TNFα, IL-1B, platelet aggregation, decrease in nitric oxide(NO) production, plaque deposition and/or normalized glycemic control.

In one embodiment, key elements of a metabolic disorder include impairedfasting glucose, impaired glucose tolerance, increased waistcircumference, increased visceral fat content, increased fasting plasmaglucose, increased fasting plasma triglycerides, decreased fasting highdensity lipoprotein level, increased blood pressure, insulin resistance,hyperinsulinemia, cardiovascular disease, arteriosclerosis, coronaryartery disease, peripheral vascular disease, cerebrovascular disease,congestive heart failure, elevated plasma norepinephrine, elevatedcardiovascular-related inflammatory factors, elevated plasma factorspotentiating vascular endothelial dysfunction, hyperlipoproteinemia,arteriosclerosis or atherosclerosis, hyperphagia, hyperglycemia,hyperlipidemia, and hypertension or high blood pressure, increasedplasma postprandial triglyceride or free fatty acid levels, increasedcellular oxidative stress or plasma indicators thereof, increasedcirculating hypercoagulative state, hepatic steatosis, hetapticsteatosis, renal disease including renal failure and renalinsufficiency.

In one embodiment of the methods of the invention, the method furthercomprises administering an additional therapeutic agent, e.g., diabetesmellitus-treating agents, diabetic complication-treating agents,antihyperlipemic agents, hypotensive or antihypertensive agents,anti-obesity agents, diuretics, chemotherapeutic agents,immunotherapeutic agents and immunosuppressive agents. In oneembodiment, the metabolic disorder is selected from the group consistingof diabetes, obesity, pre-diabetes, Metabolic Syndrome and any keyelements of a metabolic disorder. In one embodiment, a key element of ametabolic disorder is selected from the group consisting of impairedfasting glucose, impaired glucose tolerance, increased waistcircumference, increased visceral fat content, increased fasting plasmaglucose, increased fasting plasma triglycerides, decreased fasting highdensity lipoprotein level, increased blood pressure, insulin resistance,hyperinsulinemia, cardiovascular disease, arteriosclerosis, coronaryartery disease, peripheral vascular disease, cerebrovascular disease,congestive heart failure, elevated plasma norepinephrine, elevatedcardiovascular-related inflammatory factors, elevated plasma factorspotentiating vascular endothelial dysfunction, hyperlipoproteinemia,arteriosclerosis or atherosclerosis, hyperphagia, hyperglycemia,hyperlipidemia, and hypertension or high blood pressure, increasedplasma postprandial triglyceride or free fatty acid levels, increasedcellular oxidative stress or plasma indicators thereof, increasedcirculating hypercoagulative state, hepatic steatosis, hetapticsteatosis, renal disease including renal failure and renalinsufficiency.

In one embodiment of the methods of the invention, the method furthercomprises administering an additional therapeutic agent, e.g., diabetesmellitus-treating agents, diabetic complication-treating agents,antihyperlipemic agents, hypotensive or antihypertensive agents,anti-obesity agents, diuretics, chemotherapeutic agents,immunotherapeutic agents and immunosuppressive agents.

The invention provides, in another aspect, a method of identifying anagent that is effective in treating a metabolic disorder, the methodcomprising selecting an environmental influencer; identifying anenvironmental influencer capable of shifting the metabolic state of acell; and determining whether the environmental influencer is effectivein treating the metabolic disorder; thereby identifying an agent that iseffective in treating a metabolic disorder.

In one embodiment, an environmental influencer is identified as capableof shifting the metabolic state of a cell by measuring changes in anyone or more of mRNA expression, protein expression, lipid or metaboliteconcentration, levels of bioenergetic molecules, cellular energetics,mitochondrial function and mitochondrial number.

In one embodiment, the environmental influencer effective in treating ametabolic disorder is capable of reducing glucose levels or lipid levelsin a patient.

The invention provides, in still another aspect, a method of identifyinga Multidimensional Intracellular Molecule, comprising contacting a cellwith an endogenous molecule; monitoring the effect of the endogenousmolecule on a cellular microenvironment profile; and identifying anendogenous molecule that induces a change to the cellularmicroenvironment profile; thereby identifying a MultidimensionalIntracellular Molecule.

In one embodiment, the method further comprises comparing the effects ofthe endogenous molecule on the cellular microenvironment profile of adiseased cell and a normal control cell; and identifying an endogenousmolecule that differentially induces a change to the cellularmicroenvironment profile of the diseased cell as compared to the normalcontrol cell; thereby identifying a MIM.

In one embodiment, the effect on the cellular microenvironment profileis monitored by measuring a change in the level or activity of acellular molecule selected from the group consisting of mRNA, protein,lipid and metabolite.

The invention provides, in still another aspect, a method of identifyingan Epimetabolic shifter, comprising comparing molecular profiles for twoor more cells or tissues, wherein the two or more cells or tissuesdisplay differential disease states; identifying a molecule from themoleculer profiles for which a change in level correlates to the diseasestate; introducing the molecule to a cell; and evaluating the ability ofthe molecule to shift the metabolic state of a cell, wherein a moleculecapable of shifting the metabolic state of a cell is identified as anEpimetabolic shifter.

In one embodiment, the molecular profile is selected from the groupconsisting of a metabolite profile, lipid profile, protein profile orRNA profile.

In one embodiment, the molecule does not negatively effect the health orgrowth of a normal cell.

The invention provides, in another aspect, a composition comprising anagent identified according to any of the methods of the invention. Theinvention further provides, in a related aspect, a kit comprising acomposition of the invention.

The invention provides, in another aspect, a method of reducing glucoselevels in a patient comprising administering to the patient an effectiveamount of a composition of the invention. The invention provides, in arelated aspect, a method of reducing lipid levels in a patientcomprising administering to the patient an effective amount of acomposition of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Sensitivity of SK-MEL-28 to 24 hours of Q10 treatment measuredby the amount of early and late apoptotic cells.

FIG. 2: Sensitivity of SKBR3 to 24 hours of Q10 treatment measured bythe amount of early and late apoptotic cells.

FIG. 3: Sensitivity of PaCa2 to 24 hours of Q10 treatment measured bythe amount of early and late apoptotic cells.

FIG. 4: Sensitivity of PC-3 to 24 hours of Q10 treatment measured by theamount of early and late apoptotic cells.

FIG. 5: Sensitivity of HepG2 to 24 hours of Q10 treatment measured bythe amount of early and late apoptotic cells.

FIG. 6: Sensitivity of MCF-7 to 24 hours of Q10 treatment measured bythe amount of early and late apoptotic cells.

FIG. 7: Measurement of apoptotic cells upon 24 hour treatment with Q10,as measured by Apostrand ELISA method.

FIG. 8: Example gel analysis of 2-D gel electrophoresis. Spots excisedfor identification are marked.

FIG. 9: Network of interaction between proteins identified by 2-D gelelectrophoresis as being modulated by Q10 in SK-MEL-28 cells.

FIG. 10: The pentose phosphate pathway adapted from Verhoeven et al.(Am. J. Hum. Genet. 2001 68(5):1086-1092).

FIG. 11: 2-D gel of the mitochondrial enriched material of SK-MEL-28cells. Spots excised and identified by mass spectrometrycharacterization are marked.

FIG. 12: Comparative plot of the relative amounts of Q10 present inSK-MEL-28 mitochondria following the exogenous addition of 100 μM Q10into the culture medium.

FIG. 13: Apoptosis pathway mapping known processes.

FIG. 14: Western blot analysis of Bcl-x1.

FIG. 15: Western blot analysis of SK-MEL-28 sample set proved with aVimentin antibody.

FIG. 16: Western blot analysis of cell lysis from a number of celllines, evaluated with five antibodies targeting oxidativephosphorylation complexes (MitoSciences #MS601).

FIG. 17: Western blot comparison of F1-alpha levels.

FIG. 18: Western blot comparison of Q10 response with C-III-Core 2.

FIG. 19: Western blot comparison of Q10 response with C-II-30.

FIG. 20: Western blot comparison of Q10 response with C-IV-COX II.

FIG. 21: Western blot comparison of Q10 response with C-1-20 (ND6).

FIG. 22: Western blot analysis of a variety of cell types against fivemitochondrial protein.

FIG. 23: Western blot comparison of Q10 response with Complex V proteinC-V-α.

FIG. 24: Western blot comparison of Q10 response with C-III-Core 1.

FIG. 25: Western blot comparison of Q10 response with Porin (VDAC1).

FIG. 26: Western blot comparison of Q10 response with Cyclophilin D

FIG. 27: Western blot comparison of Q10 response with Cytochrome C.

FIG. 28: Theoretical model of Q10 (spheres) inserted into the lipidbinding channel of HNF4alpha (1M7W.pdb) in the Helix 10 openconformation.

FIG. 29: OCR in HDFa cells in various glucose conditions in normoxic andhypoxic conditions.

FIG. 30: OCR in HASMC cells in various glucose conditions in normoxicand hypoxic conditions.

FIG. 31: OCR values in MCF-7 breast cancer cells in the absence andpresence of 31510 and stressors.

FIG. 32: OCR values in PaCa-2 pancreatic cancer cells in the absence andpresence of 31510 and stressors.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

The term “such as” is used herein to mean, and is used interchangeably,with the phrase “such as but not limited to”.

A “patient” or “subject” to be treated by the method of the inventioncan mean either a human or non-human animal, preferably a mammal.

“Therapeutically effective amount” means the amount of a compound that,when administered to a patient for treating a disease, is sufficient toeffect such treatment for the disease. When administered for preventinga disease, the amount is sufficient to avoid or delay onset of thedisease. The “therapeutically effective amount” will vary depending onthe compound, the disease and its severity and the age, weight, etc., ofthe patient to be treated.

“Preventing” or “prevention” refers to a reduction in risk of acquiringa disease or disorder (i.e., causing at least one of the clinicalsymptoms of the disease not to develop in a patient that may be exposedto or predisposed to the disease but does not yet experience or displaysymptoms of the disease).

The term “prophylactic” or “therapeutic” treatment refers toadministration to the subject of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, i.e., it protects thehost against developing the unwanted condition, whereas if administeredafter manifestation of the unwanted condition, the treatment istherapeutic (i.e., it is intended to diminish, ameliorate or maintainthe existing unwanted condition or side effects therefrom).

The term “therapeutic effect” refers to a local or systemic effect inanimals, particularly mammals, and more particularly humans caused by apharmacologically active substance. The term thus means any substanceintended for use in the diagnosis, cure, mitigation, treatment orprevention of disease or in the enhancement of desirable physical ormental development and conditions in an animal or human. The phrase“therapeutically-effective amount” means that amount of such a substancethat produces some desired local or systemic effect at a reasonablebenefit/risk ratio applicable to any treatment. In certain embodiments,a therapeutically-effective amount of a compound will depend on itstherapeutic index, solubility, and the like. For example, certaincompounds discovered by the methods of the present invention may beadministered in a sufficient amount to produce a reasonable benefit/riskratio applicable to such treatment.

By “patient” is meant any animal (e.g., a human), including horses,dogs, cats, pigs, goats, rabbits, hamsters, monkeys, guinea pigs, rats,mice, lizards, snakes, sheep, cattle, fish, and birds.

“Metabolic pathway” refers to a sequence of enzyme-mediated reactionsthat transform one compound to another and provide intermediates andenergy for cellular functions. The metabolic pathway can be linear orcyclic.

“Metabolic state” refers to the molecular content of a particularcellular, multicellular or tissue environment at a given point in timeas measured by various chemical and biological indicators as they relateto a state of health or disease.

The term “microarray” refers to an array of distinct polynucleotides,oligonucleotides, polypeptides (e.g., antibodies) or peptidessynthesized on a substrate, such as paper, nylon or other type ofmembrane, filter, chip, glass slide, or any other suitable solidsupport.

The terms “disorders” and “diseases” are used inclusively and refer toany deviation from the normal structure or function of any part, organor system of the body (or any combination thereof). A specific diseaseis manifested by characteristic symptoms and signs, includingbiological, chemical and physical changes, and is often associated witha variety of other factors including, but not limited to, demographic,environmental, employment, genetic and medically historical factors.Certain characteristic signs, symptoms, and related factors can bequantitated through a variety of methods to yield important diagnosticinformation.

The term “expression” is used herein to mean the process by which apolypeptide is produced from DNA. The process involves the transcriptionof the gene into mRNA and the translation of this mRNA into apolypeptide. Depending on the context in which used, “expression” mayrefer to the production of RNA, protein or both.

The terms “level of expression of a gene” or “gene expression level”refer to the level of mRNA, as well as pre-mRNA nascent transcript(s),transcript processing intermediates, mature mRNA(s) and degradationproducts, or the level of protein, encoded by the gene in the cell.

The term “modulation” refers to upregulation (i.e., activation orstimulation), downregulation (i.e., inhibition or suppression) of aresponse, or the two in combination or apart. A “modulator” is acompound or molecule that modulates, and may be, e.g., an agonist,antagonist, activator, stimulator, suppressor, or inhibitor.

The term “intermediate of the coenzyme biosynthesis pathway” as usedherein, characterizes those compounds that are formed between thechemical/biological conversion of tyrosine and Acetyl-CoA to uqiquinone.Intermediates of the coenzyme biosynthesis pathway include3-hexaprenyl-4-hydroxybenzoate, 3-hexaprenyl-4,5-dihydroxybenzoate,3-hexaprenyl-4-hydroxy-5-methoxybenzoate,2-hexaprenyl-6-methoxy-1,4-benzoquinone,2-hexaprenyl-3-methyl-6-methoxy-1,4-benzoquinone,2-hexaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone,3-Octaprenyl-4-hydroxybenzoate, 2-octaprenylphenol,2-octaprenyl-6-metholxyphenol,2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinone,2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone,2-decaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone,2-decaprenyl-3-methyl-6-methoxy-1,4-benzoquinone,2-decaprenyl-6-methoxy-1,4-benzoquinone, 2-decaprenyl-6-methoxyphenol,3-decaprenyl-4-hydroxy-5-methoxybenzoate,3-decaprenyl-4,5-dihydroxybenzoate, 3-decaprenyl-4-hydroxybenzoate,4-hydroxy phenylpyruvate, 4-hydroxyphenyllactate, 4-hydroxy-benzoate,4-hydroxycinnamate and hexaprenydiphosphate.

As used herein, the phrase “anaerobic use of glucose” or “anaerobicglycolysis” refers to cellular production of energy by glycolysisfollowed by lactic acid fermentation in the cytosol. For example, manycancer cells produce energy by anaerobic glycolysis.

As used herein, the phrase “aerobic glycolysis” or “mitochondrialoxidative phosphorylation” refers to cellular production of energy byglycolysis followed by oxidation of pyruvate in mitochondria.

As used herein, the phrase “capable of blocking anaerobic use of glucoseand augmenting mitochondrial oxidative phosphorylation” refers to theability of an environmental influencer (e.g., an epitmetabolic shifter)to induce a shift or change in the metabolic state of a cell fromanaerobic glycolysis to aerobic glycolysis or mitochondrial oxidativephosphorylation.

Reference will now be made in detail to preferred embodiments of theinvention. While the invention will be described in conjunction with thepreferred embodiments, it will be understood that it is not intended tolimit the invention to those preferred embodiments. To the contrary, itis intended to cover alternatives, modifications, and equivalents as maybe included within the spirit and scope of the invention as defined bythe appended claims.

II. Environmental Influencers

The present invention provides methods of treating metabolic disordersby administration of an Environmental influencer. “Environmentalinfluencers” (Env-influencers) are molecules that influence or modulatethe disease environment of a human in a beneficial manner allowing thehuman's disease environment to shift, reestablish back to or maintain anormal or healthy environment leading to a normal state. Env-influencersinclude both Multidimensional Intracellular Molecules (MIMs) andEpimetabolic shifters (Epi-shifters) as defined below.

1. Multidimensional Intracellular Molecule (MIM)

The term “Multidimensional Intracellular Molecule (MIM)”, is an isolatedversion or synthetically produced version of an endogenous molecule thatis naturally produced by the body and/or is present in at least one cellof a human. A MIM is characterized by one or more, two or more, three ormore, or all of the following functions. MIMs are capable of entering acell, and the entry into the cell includes complete or partial entryinto the cell, as long as the biologically active portion of themolecule wholly enters the cell. MIMs are capable of inducing a signaltransduction and/or gene expression mechanism within a cell. MIMs aremultidimensional in that the molecules have both a therapeutic and acarrier, e.g., drug delivery, effect. MIMs also are multidimensional inthat the molecules act one way in a disease state and a different way ina normal state. For example, in the case of CoQ-10, administration ofCoQ-10 to a melanoma cell in the presence of VEGF leads to a decreasedlevel of Bc12 which, in turn, leads to a decreased oncogenic potentialfor the melanoma cell. In contrast, in a normal fibroblast,co-administration of CoQ-10 and VEFG has no effect on the levels ofBc12. Preferably, MIMs selectively act in cells of a disease state, andhave substantially no effect in (matching) cells of a normal state.Preferably, MIMs selectively renders cells of a disease state closer inphenotype, metabolic state, genotype, mRNA/protein expression level,etc. to (matching) cells of a normal state.

In one embodiment, a MIM is also an epi-shifter. In another embodiment,a MIM is not an epi-shifter. The skilled artisan will appreciate that aMIM of the invention is also intended to encompass a mixture of two ormore endogenous molecules, wherein the mixture is characterized by oneor more of the foregoing functions. The endogenous molecules in themixture are present at a ratio such that the mixture functions as a MIM.

MIMs can be lipid based or non-lipid based molecules. Examples of MIMsinclude, but are not limited to, CoQ10, acetyl Co-A, palmityl Co-A,L-carnitine, amino acids such as, for example, tyrosine, phenylalanine,and cysteine. In one embodiment, the MIM is a small molecule. In oneembodiment of the invention, the MIM is not CoQ10. MIMs can be routinelyidentified by one of skill in the art using any of the assays describedin detail herein.

In some embodiments, MIMs include compounds in the Vitamin B family, ornucleosides, mononucleotides or dinucleotides that comprise a compoundin the Vitamin B family. Compounds in the vitamin B family include, forexample, thiamine (vitamin B1), niacin (also known as nicotinic acid orVitamin B3), or pyridoxine (vitamin B6) as well as provitamins such aspanthenol (provitamin B5). In some embodiments, the MIM is selected fromthiamine, niacin and pyridoxine. Nucleosides, mononucleotides ordinucleotides that comprise a compound in the vitamin B family include,for example, nucleosides, mononucleotides or dinucleotides which includean adenine or a niacin (nicotinic acid) molecule. In some embodiments,the MIM is selected from adenosine, adenosine diphosphate (ADP), flavinadenosine dinucleotide (FAD, which comprises parts of vitamin B2 andADP) and nicotinic acid dinucleotide.

In other embodiments, the MIMs include amino acids. Examples of aminoacids include, for example, tyrosine (e.g., L-tyrosine), cysteine,phenylalanine (e.g., L-phenylalanine) and alanine. In some embodiments,the amino acid is phenylalanine or alanine. In some embodiments, theMIMs include amino acid derivatives such as 4-hydroxyphenylpyruvate oracetylglycine.

In some embodiment, the MIM is a glucose analog, e.g., a glucosemolecule wherein one —OH or —CH₂OH substituent has been replaced with a—COOH, a —COO⁻ or an —NH₂ substituent. Examples of glucose analogsinclude glucosamine, glucuronic acid, glucuronide and glucuronate.

In some embodiments, the MIM is selected from compounds of formula (I):

wherein

n is an integer of 0 or 1;

R¹, R², R³ and R⁴, when present, are each independently selected fromhydrogen and hydroxyl or R¹ and R² are taken together with the carbon onwhich they are attached to form a carbonyl (C═O) group;

W is —COOH or —N(CH₃)₃ ⁺; and

X is hydrogen, a negative charge or a alkali metal cation, such as Na⁺or.

It is to be understood that when n is 0, the CHR³ group is bonded to theW substituent.

In some embodiments, W is —N(CH₃)₃ ⁺. In some embodiments, the MIM is acarnitine, such as L-carnitine.

In some embodiments, the MIM is a dicarboxylic acid. In someembodiments, W is —COOH. In some embodiments, R³ is hydrogen. In someembodiments, n is 0. In some embodiments, R¹ and R² are eachindependently hydrogen. In some embodiments, W is —COOH, R³ is hydrogen,n is 0 and R¹ and R² are each independently hydrogen. In someembodiments, n is 1. In some embodiments R¹ and R² are taken togetherwith the carbon on which they are attached to form a carbonyl (C═O)group. In some embodiments, R⁴ is hydrogen. In some embodiments, R⁴ ishydroxyl. In some embodiments, W is —COOH, R³ is hydrogen, n is 1 and R¹and R² are taken together with the carbon on which they are attached toform a carbonyl (C═O) group.

In some embodiments, the MIM is an intermediate of the Krebs Cycle, theexcess of which drives the Krebs Cycle towards productive oxidativephosphorylation. Exemplary Krebs Cycle intermediates that are MIMsinclude succinic acid or succinate, malic acid or malate, andα-ketoglutaric acid or α-ketoglutarate.

In some embodiments, the MIM is a building block of CoQ10, which has thefollowing structure:

Thus, building blocks of CoQ10 include, but are not limited to,phenylalanine, tyrosine, 4-hydroxyphenylpyruvate, phenylacetate,3-methoxy-4-hydroxymandelate, vanillic acid, 4-hydroxybenzoate,mevalonic acid, farnesyl, 2,3-dimethoxy-5-methyl-p-benzoquinone, as wellas the corresponding acids or ions thereof. In some embodiments, the MIMis selected from phenylalanine, tyrosine, 4-hydroxyphenylpyruvate,phenylacetate and 4-hydroxybenzoate.

(i) Methods of Identifying MIMS

The present invention provides methods for identifying a MIM. Methodsfor identifying a MIM involve, generally, the exogenous addition to acell of an endogenous molecule and evaluating the effect on the cell,e.g., the cellular microenvironment profile, that the endogenousmolecule provides. Effects on the cell are evaluated at one or more ofthe cellular, mRNA, protein, lipid, and/or metabolite level to identifyalterations in the cellular microenvironment profile. In one embodiment,the cells are cultured cells, e.g., in vitro. In one embodiment, thecells are present in an organism. The endogenous molecule may be addedto the cell at a single concentration or may be added to the cell over arange of concentrations. In one embodiment, the endogenous molecule isadded to the cells such that the level of the endogenous molecule in thecells is elevated (e.g., is elevated by 1.1 fold, 1.2 fold, 1.3 fold,1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2.0 fold,3.0 fold, 4.0 fold, 5.0 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30fold, 35 fold, 40 fold, 45 fold, 50 fold or greater) as compared to thelevel of the endogenous molecule in a control, untreated cell.

Molecules that induce a change in the cell as detected by alterationsin, for example, any one or more of morphology, physiology, and/orcomposition (e.g., mRNA, protein, lipid, metabolite) may be evaluatedfurther to determine if the induced changes to the cellularmicroenvironment profile are different between a disease cellular stateand a normal cellular state. Cells (e.g., cell culture lines) of diversetissue origin, cell type, or disease state may be evaluated forcomparative evaluation. For example, changes induced in the cellularmicroenvironment profile of a cancer cell may be compared to changesinduced to a non-cancerous or normal cell. An endogenous molecule thatis observed to induce a change in the microenvironment profile of a cell(e.g., induces a change in the morphology, physiology and/orcomposition, e.g., mRNA, protein, lipid or metabolite, of the cell)and/or to differentially (e.g., preferentially) induce a change in themicroenvironment profile of a diseased cell as compared to a normalcell, is identified as a MIM.

MIMs of the invention may be lipid based MIMs or non-lipid based MIMs.Methods for identifying lipid based MIMs involve the above-describedcell based methods in which a lipid based endogenous molecule isexogenously added to the cell. In a preferred embodiment, the lipidbased endogenous molecule is added to the cell such that the level ofthe lipid based endogenous molecule in the cell is elevated. In oneembodiment, the level of the lipid based endogenous molecule is elevatedby 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold,1.8 fold, 1.9 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold, 10 fold, 15fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold orgreater as compared to the level in an untreated control cell.Formulation and delivery of the lipid based molecule to the cell isdependent upon the properties of each molecule tested, but many methodsare known in the art. Examples of formulation and delivery of lipidbased molecules include, but are not limited to, solubilization byco-solvents, carrier molecules, liposomes, dispersions, suspensions,nanoparticle dispersions, emulsions, e.g., oil-in-water or water-in-oilemulsions, multiphase emulsions, e.g., oil-in-water-in-oil emulsions,polymer entrapment and encapsulation. The delivery of the lipid basedMIM to the cell can be confirmed by extraction of the cellular lipidsand quantification of the MIM by routine methods known in the art, suchas mass spectrometry.

Methods for identifying non-lipid based MIMs involve the above-describedcell based methods in which a non-lipid based endogenous molecule isexogenously added to the cell. In a preferred embodiment, the non-lipidbased endogenous molecule is added to the cell such that the level ofthe non-lipid based endogenous molecule in the cell is elevated. In oneembodiment, the level of the non-lipid based endogenous molecule iselevated by 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold,1.7 fold, 1.8 fold, 1.9 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold, 10fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50fold or greater as compared to the level in an untreated control cell.Formulation and delivery of the non-lipid based molecule to the cell isdependent upon the properties of each molecule tested, but many methodsare known in the art.

Examples of formulations and modes of delivery of non-lipid basedmolecules include, but are not limited to, solubilization byco-solvents, carrier molecules, active transport, polymer entrapment oradsorption, polymer grafting, liposomal encapsulation, and formulationwith targeted delivery systems. The delivery of the non-lipid based MIMto the cell may be confirmed by extraction of the cellular content andquantification of the MIM by routine methods known in the art, such asmass spectrometry.

2. Epimetabolic Shifters (Epi-Shifters)

As used herein, an “epimetabolic shifter” (epi-shifter) is a molecule(endogenous or exogenous) that modulates the metabolic shift from ahealthy (or normal) state to a disease state and vice versa, therebymaintaining or reestablishing cellular, tissue, organ, system and/orhost health in a human. Epi-shifters are capable of effectuatingnormalization in a tissue microenvironment. For example, an epi-shifterincludes any molecule which is capable, when added to or depleted from acell, of affecting the microenvironment (e.g., the metabolic state) of acell. The skilled artisan will appreciate that an epi-shifter of theinvention is also intended to encompass a mixture of two or moremolecules, wherein the mixture is characterized by one or more of theforegoing functions. The molecules in the mixture are present at a ratiosuch that the mixture functions as an epi-shifter. Examples ofepi-shifters include, but are not limited to, coQ-10; vitamin D3; ECMcomponents such as fibronectin; immunomodulators, such as TNFa or any ofthe interleukins, e.g., IL-5, IL-12, IL-23; angiogenic factors; andapoptotic factors.

In some embodiments, the epi-shifter is an enzyme, such as an enzymethat either directly participates in catalyzing one or more reactions inthe Krebs Cycle, or produces a Krebs Cycle intermediate, the excess ofwhich drive the Krebs Cycle. In some embodiments, the enzyme is anenzyme of the non-oxidative phase of the pentose phosphate pathway, suchas transaldolase, or transketolase. In other embodiments, the enzyme isa component enzyme or enzyme complex that facilitates the Krebs Cycle,such as a synthase or a ligase. Exemplary enzymes include succinyl CoAsynthase (Krebs Cycle enzyme) or pyruvate carboxylase (a ligase thatcatalyzes the reversible carboxylation of pyruvate to form oxaloacetate(OAA), a Krebs Cycle intermediate).

In some embodiments, the epi-shifter is a building block of CoQ10.Building blocks of CoQ10 include, but are not limited to, phenylalanine,tyrosine, 4-hydroxyphenylpyruvate, phenylacetate,3-methoxy-4-hydroxymandelate, vanillic acid, 4-hydroxybenzoate,mevalonic acid, farnesyl, 2,3-dimethoxy-5-methyl-p-benzoquinone, as wellas the corresponding acids or ions thereof. In some embodiments, theepi-shifter is selected from phenylalanine, tyrosine,4-hydroxyphenylpyruvate, phenylacetate and 4-hydroxybenzoate.

In some embodiments, the epi-shifter is a compound in the Vitamin Bfamily. Compounds in the vitamin B family include, for example,riboflavin (vitamin B2), or analogs thereof. Epi-shifters also includeany analogs or pro-drugs that may be metabolized in vivo to any of theendogenous MIMs, such as those described herein.

In one embodiment, the epi-shifter also is a MIM. In one embodiment, theepi-shifter is not CoQ10. Epi-shifters can be routinely identified byone of skill in the art using any of the assays described in detailherein.

(i) Methods of Identifying Epi-Shifters

Epimetabolic shifters (epi-shifter) are molecules capable of modulatingthe metabolic state of a cell, e.g., inducing a metabolic shift from ahealthy (or normal) state to a disease state and vice versa, and arethereby capable of maintaining or reestablishing cellular, tissue,organ, system and/or host health in a human. Epi-shifters of theinvention thus have utility in the diagnostic evaluation of a diseasedstate. Epi-shifters of the invention have further utility in therapeuticapplications, wherein the application or administration of theepi-shifter (or modulation of the epi-shifter by other therapeuticmolecules) effects a normalization in a tissue microenvironment and thedisease state.

The identification of an epimetabolic shifter involves, generally,establishing a molecular profile, e.g., of metabolites, lipids, proteinsor RNAs (as individual profiles or in combination), for a panel of cellsor tissues that display differential disease states, progression, oraggressiveness A molecule from the profile(s) for which a change inlevel (e.g., an increased or decreased level) correlates to the diseasestate, progression or aggressiveness is identified as a potentialepi-shifter.

In one embodiment, an epi-shifter is also a MIM. Potential epi-shiftersmay be evaluated for their ability to enter cells upon exogenousaddition to a cell by using any number of routine techniques known inthe art, and by using any of the methods described herein. For example,entry of the potential epi-shifter into a cell may be confirmed byextraction of the cellular content and quantification of the potentialepi-shifter by routine methods known in the art, such as massspectrometry. A potential epi-shifter that is able to enter a cell isthereby identified as a MIM.

To identify an epi-shifter, a potential epi-shifter is next evaluatedfor the ability to shift the metabolic state of a cell. The ability of apotential epi-shifters to shift the metabolic state of the cellmicroenvironment is evaluated by introducing (e.g., exogenously adding)to a cell a potential epi-shifter and monitoring in the cell one or moreof: changes in gene expression (e.g., changes in mRNA or proteinexpression), concentration changes in lipid or metabolite levels,changes in bioenergetic molecule levels, changes in cellular energetics,and/or changes in mitochondrial function or number. Potentialepi-shifters capable of shifting the metabolic state of the cellmicroenvironment can be routinely identified by one of skill in the artusing any of the assays described in detail herein. Potentialepi-shifters are further evaluated for the ability to shift themetabolic state of a diseased cell towards a normal healthy state (orconversely, for the ability to shift the metabolic state of a normalcell towards a diseased state). A potential epi-shifter capable ofshifting the metabolic state of a diseased cell towards a normal healthystate (or of shifting the metabolic state of healthy normal cell towardsa diseased state) is thus identified as an Epi-shifter. In a preferredembodiment, the epi-shifter does not negatively impact the health and/orgrowth of normal cells.

Epimetabolic shifters of the invention include, but are not limited to,small molecule metabolites, lipid-based molecules, and proteins andRNAs. To identify an epimetabolic shifter in the class of small moleculeendogenous metabolites, metabolite profiles for a panel of cells ortissues that display differential disease states, progression, oraggressiveness are established. The metabolite profile for each cell ortissue is determined by extracting metabolites from the cell or tissueand then identifying and quantifying the metabolites using routinemethods known to the skilled artisan, including, for example,liquid-chromatography coupled mass spectrometry or gas-chromatographycouple mass spectrometry methods. Metabolites for which a change inlevel (e.g., an increased or decreased level) correlates to the diseasestate, progression or aggressiveness, are identified as potentialepi-shifters.

To identify epimetabolic shifters in the class of endogenous lipid-basedmolecules, lipid profiles for a panel of cells or tissues that displaydifferential disease states, progression, or aggressiveness areestablished. The lipid profile for each cell or tissue is determined byusing lipid extraction methods, followed by the identification andquantitation of the lipids using routine methods known to the skilledartisan, including, for example, liquid-chromatography coupled massspectrometry or gas-chromatography couple mass spectrometry methods.Lipids for which a change in level (e.g., an increase or decrease inbulk or trace level) correlates to the disease state, progression oraggressiveness, are identified as potential epi-shifters.

To identify epimetabolic shifters in the class of proteins and RNAs,gene expression profiles for a panel of cells or tissues that displaydifferential disease states, progression, or aggressiveness areestablished. The expression profile for each cell or tissue isdetermined at the mRNA and/or protein level(s) using standard proteomic,mRNA array, or genomic array methods, e.g., as described in detailherein. Genes for which a change in expression (e.g., an increase ordecrease in expression at the mRNA or protein level) correlates to thedisease state, progression or aggressiveness, are identified aspotential epi-shifters.

Once the molecular profiles described above are established (e.g., forsoluble metabolites, lipid-based molecules, proteins, RNAs, or otherbiological classes of composition), cellular and biochemical pathwayanalysis is carried out to elucidate known linkages between theidentified potential epi-shifters in the cellular environment.

This information obtained by such cellular and/or biochemical pathwayanalysis may be utilized to categorize the pathways and potentialepi-shifters.

The utility of an Epi-shifter to modulate a disease state can be furtherevaluated and confirmed by one of skill in the art using any number ofassays known in the art or described in detail herein. The utility of anEpi-shifter to modulate a disease state can be evaluated by directexogenous delivery of the Epi-shifter to a cell or to an organism. Theutility of an Epi-shifter to modulate a disease state can alternativelybe evaluated by the development of molecules that directly modulate theEpi-shifter (e.g., the level or activity of the Epi-shifter). Theutility of an Epi-shifter to modulate a disease state can also beevaluated by the development of molecules that indirectly modulate theEpi-shifter (e.g., the level or activity of the Epi-shifter) byregulating other molecules, such as genes (e.g., regulated at the RNA orprotein level), placed in the same pathway as the Epi-shifter.

The Epimetabolomic approach described herein facilitates theidentification of endogenous molecules that exist in a cellularmicroenvironment and the levels of which are sensed and controlledthrough genetic, mRNA, or protein-based mechanisms. The regulationresponse pathways found in normal cells that are triggered by anEpi-shifter of the invention may provide a therapeutic value in amisregulated or diseased cellular environment. In addition, theepimetabolic approach described herein identifies epi-shifters that mayprovide a diagnostic indication for use in clinical patient selection, adisease diagnostic kit, or as a prognostic indicator.

In certain embodiments, the MIMS and Epi-shifters disclosed hereinexclude those that are conventionally used as a dietary supplement. Incertain embodiments, these MIMS and/or Epi-shifter that are disclosedherein are of pharmaceutical grade. In certain embodiments, the MIMSand/or Epi-shifter of pharmaceutical grade has a purity between about95% and about 100% and include all values between 95% and 100%. Incertain embodiments, the purity of the MIMS and/or Epi-shifter is 95%,96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, 99.9%, 99.9 or 100%. In certain embodiments, the MIMS and/orEpi-shifter is free of endotoxins. In other embodiments, the MIMS and/orEpi-shifter is free of foreign protein materials. In certainembodiments, the MIMS and/or Epi-shifter is CoQ10.

III. Assays Useful for identifying MIMs/Epi-Shifters

Techniques and methods of the present invention employed to separate andidentify molecules and compounds of interest include but are not limitedto: liquid chromatography (LC), high-pressure liquid chromatography(HPLC), mass spectroscopy (MS), gas chromatography (GC), liquidchromatography/mass spectroscopy (LC-MS), gas chromatography/massspectroscopy (GC-MS), nuclear magnetic resonance (NMR), magneticresonance imaging (MRI), Fourier Transform InfraRed (FT-IR), andinductively coupled plasma mass spectrometry (ICP-MS). It is furtherunderstood that mass spectrometry techniques include, but are notlimited to, the use of magnetic-sector and double focusing instruments,transmission quadrapole instruments, quadrupole ion-trap instruments,time-of-flight instruments (TOF), Fourier transform ion cyclotronresonance instruments (FT-MS) and matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Quantification of Bioenergetic Molecule Levels:

Environmental influencers (e.g., MIMs or Epi-shifters) may be identifiedby changes in cellular bioenergetic molecule levels (e.g., ATP,pyruvate, ADP, NADH, NAD, NADPH, NADP, acetylCoA, FADH2) of cells towhich a candidate epi-shifter has been applied. Exemplary assays ofbioenergetic molecule levels use colorometric, fluorescence, and/orbioluminescent-based methods. Examples of such assays are providedbelow.

Levels of ATP within cells can be measured with a number of assays andsystems known in the art. For example, in one system, cytoplasmic ATPreleased from lysed cells reacts with luciferin and the enzymeluciferase to produce light. This bioluminescence is measured by abioluminometer and the intracellular ATP concentration of the lysedcells can be calculated (EnzyLight™ ATP Assay Kit (EATP-100), BioAssaySystems, Hayward, Calif.). In another system, for example, both ATP andits dephosphorylated form, ADP, are calculated via bioluminescence;after ATP levels are calculated, ADP is transformed into ATP and thendetected and calculated using the same luciferase system (ApoSENSOR™ADP/ATP Ratio Assay Kit, BioVision Inc., Mountain View, Calif.).

Pyruvate is an important intermediate in cellular metabolic pathways.Pyruvate may be converted into carbohydrate via gluconeogenesis,converted into fatty acid or metabolized via acetyl CoA, or convertedinto alanine or ethanol, depending upon the metabolic state of a cell.Thus detection of pyruvate levels provides a measure of the metabolicactivity and state of a cell sample. One assay to detect pyruvate, forexample, uses both a colorimetric and fluorimetric to detect pyruvateconcentrations within different ranges (EnzyChrom™ Pyruvate Assay Kit(Cat# EPYR-100), BioAssay Systems, Hayward, Calif.).

Environmental influencers (e.g., MIMs or Epi-shifters) may influence theprocess of oxidative phosphorylation carried out by mitochondria incells, which are involved in the generation and maintenance ofbioenergetic molecules in cells. In addition to assays that detectchanges in cellular energetics in cell cultures and samples directly(described below), assays exist that detect and quantify the effects ofcompounds on discrete enzymes and complexes of mitochondria in cells.For example, the MT-OXC MitoTox™ Complete OXPHOS Activity Assay(MitoSciences Inc., Eugene, Oreg.) can detect and quantify the effectsof compounds applied directly to complexes Ito V extracted frommitochondria. Assays for the detection and quantification of effects onindividual mitochondrial complexes such as NADH dehydrogenase (ComplexI), cytochrome c oxidase (Complex IV) and ATP synthase (Complex V) arealso available (MitoSciences Inc., Eugene, Oreg.).

Measurement of Cellular Energetics:

Environmental influencers (e.g., MIMs or Epi-shifters) may also beidentified by changes in cellular energetics. One example of themeasurement of cellular energetics are the real-time measures of theconsumption of molecular oxygen and/or the change in pH of the media ofa cell culture. For example, the ability of a potential epi-shifter tomodulate the metabolic state of a cell may be analyzed using, forexample, the XF24 Analyzer (Seahorse, Inc.). This technology allows forreal time detection of oxygen and pH changes in a monolayer of cells inorder to evaluate the bioenergetics of a cell microenvironment. The XF24Analyzer measures and compares the rates of oxygen consumption (OCR),which is a measure of aerobic metabolism, and extracellularacidification (ECAR), which is a measure of glycolysis, both keyindicators of cellular energetics.

Measurement of Oxidative Phosphorylation and Mitochondrial Function

Oxidative Phosphorylation is a process by which ATP is generated via theoxidation of nutrient compounds, carried out in eukaryotes via proteincomplexes embedded in the membranes of mitochondria. As the primarysource of ATP in the cells of most organisms, changes in oxidativephosphorylation activity can strongly alter metabolism and energybalance within a cell. In some embodiments of the invention,environmental influencers (e.g., MIMs or Epi-shifters) may be detectedand/or identified by their effects on oxidative phosphorylation. In someembodiments, environmental influencers (e.g., MIMs or Epi-shifters) maybe detected and/or identified by their effects on specific aspects ofoxidative phosphorylation, including, but not limited to, the electrontransport chain and ATP synthesis.

The membrane-embedded protein complexes of the mitochrondria that carryout processes involved in oxidative phosphorylation perform specifictasks and are numbered I, II, III and IV. These complexes, along withthe trans-inner membrane ATP synthase (also known as Complex V), are thekey entities involved in the oxidative phosphorylation process. Inaddition to assays that can examine the effects of environmentalinfluencers (e.g., MIMs or Epi-shifters) on mitochondrial function ingeneral and the oxidative phosphorylation process in particular, assaysare available that can be used to examine the effects of an epi-shifteron an individual complex separately from other complexes.

Complex I, also known as NADH-coenzyme Q oxidoreductase or NADHdehydrogenase, is the first protein in the electron transport chain. Insome embodiments, the detection and quantification of the effect of anepi-shifter on the production of NAD by Complex I may be performed. Forexample, the complex can be immunocaptured from a sample in a 96-wellplate; the oxidation of NADH to NAD takes place concurrently with thereduction of a dye molecule which has an increased absorbance at 450 nM(Complex I Enzyme Activity Microplate Assay Kit, MitoSciences Inc.,Eugene, Oreg.).

Complex IV, also known as cytochrome c oxidase (COX), is the lastprotein in the electron transport chain. In some embodiments, thedetection and quantification of the effect of an epi-shifter on theoxidation of cytochrome c and the reduction of oxygen to water byComplex IV may be performed. For example, COX can be immunocaptured in amicrowell plate and the oxidation of COX measured with a colorimetricassay (Complex IV Enzyme Activity Microplate Assay Kit, MitoSciencesInc., Eugene, Oreg.).

The final enzyme in the oxidative phosphorylation process is ATPsynthase (Complex V), which uses the proton gradient created by theother complexes to power the synthesis of ATP from ADP. In someembodiments, the detection and quantification of the effect of anepi-shifter on the activity of ATP synthase may be performed. Forexample, both the activity of ATP synthase and the amount of ATPsynthase in a sample may be measured for ATP synthase that has beenimmunocaptured in a microwell plate well. The enzyme can also functionas an ATPase under certain conditions, thus in this assay for ATPsynthase activity, the rate at which ATP is reduced to ADP is measuredby detecting the simultaneous oxidation of NADH to NAD⁺. The amount ofATP is calculated using a labeled antibody to ATPase (ATP synthaseDuplexing (Activity+Quantity) Microplate Assay Kit, MitoSciences Inc.,Eugene, Oreg.). Additional assays for oxidative phosphorylation includeassays that test for effects on the activity of Complexes II and III.For example, the MT-OXC MitoTox™ Complete OXPHOS System (MitoSciencesInc., Eugene, Oreg.) can be used to evaluate effects of a compound onComplex II and III as well as Complex I, IV and V, to provide data onthe effects of a compound on the entire oxidative phosphorylationsystem.

As noted above, real-time observation of intact cell samples can be madeusing probes for changes in oxygen consumption and pH in cell culturemedia. These assays of cell energetics provide a broad overview ofmitochondrial function and the effects of potential environmentalinfluencers (e.g., MIMs or Epi-shifters) on the activity of mitochondriawithin the cells of the sample.

Environmental influencers (e.g., MIMs or Epi-shifters) may also affectmitochondrial permeability transition (MPT), a phenomena in which themitochondrial membranes experience an increase in permeability due tothe formation of mitochondrial permeability transition pores (MPTP). Anincrease in mitochondrial permeability can lead to mitochondrialswelling, an inability to conduct oxidative phosphorylation and ATPgeneration and cell death. MPT may be involved with induction ofapoptosis. (See, for example, Halestrap, A. P., Biochem. Soc. Trans.34:232-237 (2006) and Lena, A. et al. Journal of Translational Med.7:13-26 (2009), hereby incorporated by reference in their entirety.)

In some embodiments, the detection and quantification of the effect ofan environmental influencer (e.g., MIM or epi-shifter) on the formation,discontinuation and/or effects of MPT and MPTPs are measured. Forexample, assays can detect MPT through the use of specialized dyemolecules (calcein) that are localized within the inner membranes ofmitochondria and other cytosolic compartments. The application ofanother molecule, CoCl₂, serves to squelch the fluorescence of thecalcein dye in the cytosol. CoCl₂ cannot access, however, the interiorof the mitochondria, thus the calcein fluorescence in the mitochondriais not squelched unless MPT has occurred and CoCl₂ can access theinterior of the mitochondra via MPTPs. Loss of mitochondrial-specificfluorescence signals that MPT has occurred. Flow cytometry can be usedto evaluate cellular and organelle fluorescence (MitoProbe™ TransitionPore Assay Kit, Molecular Probes, Eugene, Oreg.). Additional assaysutilize a fluorescence microscope for evaluating experimental results(Image-iT LIVE Mitochondrial Transition Pore Assay Kit, MolecularProbes, Eugene, Oreg.).

Measurement of Cellular Proliferation and Inflammation

In some embodiments of the invention, environmental influencers (e.g.,MIMs or Epi-shifters) may be identified and evaluated by their effectson the production or activity of molecules associated with cellularproliferation and/or inflammation. These molecules include, but are notlimited to, cytokines, growth factors, hormones, components of theextra-cellular matrix, chemokines, neuropeptides, neurotransmitters,neurotrophins and other molecules involved in cellular signaling, aswell as intracellular molecules, such as those involved in signaltransduction.

Vascular endothelial growth factor (VEGF) is a growth factor with potentangiogenic, vasculogenic and mitogenic properties. VEGF stimulatesendothelial permeability and swelling and VEGF activity is implicated innumerous diseases and disorders, including rheumatoid arthritis,metastatic cancer, age-related macular degeneration and diabeticretinopathy.

In some embodiments of the invention, an environmental influencer (e.g.,MIM or Epi-shifter) may be identified and characterized by its effectson the production of VEGF. For example, cells maintained in hypoxicconditions or in conditions mimicking acidosis will exhibit increasedVEGF production. VEGF secreted into media can be assayed using an ELISAor other antibody-based assays, using available anti-VEGF antibodies(R&D Systems, Minneapolis, Minn.). In some embodiments of the invention,an Epi-shifter may be identified and/or characterized based on itseffect(s) on the responsiveness of cells to VEGF and/or based on itseffect(s) on the expression or activity of the VEGF receptor.

Implicated in both healthy immune system function as well as inautoimmune diseases, tumor necrosis factor (TNF) is a key mediator ofinflammation and immune system activation. In some embodiments of theinvention, an Epi-shifter may be identified and characterized by itseffects on the production or the activity of TNF. For example, TNFproduced by cultured cells and secreted into media can be quantified viaELISA and other antibody-based assays known in the art. Furthermore, insome embodiments an environmental influencer may be identified andcharacterized by its effect(s) on the expression of receptors for TNF(Human TNF RI Duoset, R&D Systems, Minneapolis, Minn.).

The components of the extracellular matrix (ECM) play roles in both thestructure of cells and tissues and in signaling processes. For example,latent transforming growth factor beta binding proteins are ECMcomponents that create a reservoir of transforming growth factor beta(TGFβ) within the ECM. Matrix-bound TGFβ can be released later duringthe process of matrix remodeling and can exert growth factor effects onnearby cells (Dallas, S. Methods in Mol. Biol. 139:231-243 (2000)).

In some embodiments, an environmental influencer (e.g., MIM orEpi-shifter) may be identified or characterized by its effect(s) on thecreation of ECM by cultured cells. Researchers have developed techniqueswith which the creation of ECM by cells, as well as the composition ofthe ECM, can be studied and quantified. For example, the synthesis ofECM by cells can be evaluated by embedding the cells in a hydrogelbefore incubation. Biochemical and other analyses are performed on theECM generated by the cells after cell harvest and digestion of thehydrogel (Strehin, I. and Elisseeff, J. Methods in Mol. Bio. 522:349-362(2009)).

In some embodiments, the effect of environmental influencer (e.g., MIMor epi-shifter) on the production, status of or lack of ECM or one ofits components in an organism may be identified or characterized.Techniques for creating conditional knock-out (KO) mice have beendeveloped that allow for the knockout of particular ECM genes only indiscrete types of cells or at certain stages of development (Brancaccio,M. et al. Methods in Mol. Bio. 522:15-50 (2009)). The effect of theapplication or administration of an epi-shifter or potential epi-shifteron the activity or absence of a particular ECM component in a particulartissue or at a particular stage of development may thus be evaluated.

Measurement of Plasma Membrane Integrity and Cell Death

Environmental influencers (e.g., MIMs or Epi-shifters) may be identifiedby changes in the plasma membrane integrity of a cell sample and/or bychanges in the number or percentage of cells that undergo apoptosis,necrosis or cellular changes that demonstrate an increased or reducedlikelihood of cell death.

An assay for lactate dehydrogenase (LDH) can provide a measurement ofcellular status and damage levels. LDH is a stable and relativelyabundant cytoplasmic enzyme. When plasma membranes lose physicalintegrity, LDH escapes to the extracellular compartment. Higherconcentrations of LDH correlate with higher levels of plasma membranedamage and cell death. Examples of LDH assays include assays that use acolorimetric system to detect and quantify levels of LDH in a sample,wherein the reduced form of a tetrazolium salt is produced via theactivity of the LDH enzyme (QuantiChrom™ Lactate Dehydrogenase Kit(DLDH-100), BioAssay Systems, Hayward, Calif.; LDH CytotoxicityDetection Kit, Clontech, Mountain View, Calif.).

Apoptosis is a process of programmed cell death that may have a varietyof different initiating events. A number of assays can detect changes inthe rate and/or number of cells that undergo apoptosis. One type ofassay that is used to detect and quantify apoptosis is a capase assay.Capases are aspartic acid-specific cysteine proteases that are activatedvia proteolytic cleavage during apoptosis. Examples of assays thatdetect activated capases include PhiPhiLux® (OncoImmunin, Inc.,Gaithersburg, Md.) and Caspase-Glo® 3/7 Assay Systems (Promega Corp.,Madison, Wis.). Additional assays that can detect apoptosis and changesin the percentage or number of cells undergoing apoptosis in comparativesamples include TUNEL/DNA fragmentation assays. These assays detect the180 to 200 base pair DNA fragments generated by nucleases during theexecution phase of apoptosis. Exemplary TUNEL/DNA fragmentation assaysinclude the In Situ Cell Death Detection Kit (Roche Applied Science,Indianapolis, Ind.) and the DeadEnd™ Colorimetric and Fluorometric TUNELSystems (Promega Corp., Madison, Wis.).

Some apoptosis assays detect and quantify proteins associated with anapoptotic and/or a non-apoptotic state. For example, the MultiTox-FluorMultiplex Cytotoxicity Assay (Promega Corp., Madison, Wis.) uses asingle substrate, fluorimetric system to detect and quantify proteasesspecific to live and dead cells, thus providing a ratio of living cellsto cells that have undergone apoptosis in a cell or tissue sample.

Additional assays available for detecting and quantifying apoptosisinclude assays that detect cell permeability (e.g., APOPercentage™APOPTOSIS Assay, Biocolor, UK) and assays for Annexin V (e.g., AnnexinV-Biotin Apoptosis Detection Kit, BioVision Inc., Mountain View,Calif.).

IV. Treatment of Metabolic Disorders

In some embodiments, the compounds of the present invention, e.g., theenvironmental influencers, e.g., MIMs or epi-shifters, described herein,may be used to treat a Coenzyme Q10 responsive state in a subject inneed thereof. The language “Coenzyme Q10 responsive state,” or “CoQ10responsive state,” includes diseases, disorders, states and/orconditions which can be treated, prevented, or otherwise ameliorated bythe administration of Coenzyme Q10. Without wishing to be bound by anyparticular theory, and as described further herein, it is believed thatCoQ10 functions, at least partially, by inducing a metabolic shift tothe cell microenvironment, such as a shift towards the type and/or levelof oxidative phosphorylation in normal state cells. Accordingly, in someembodiments, CoQ10 responsive states are states that arise from analtered metabolism of cell microenvironment. In one embodiment, theCoQ10 responsive disorder is a metabolic disorder. Coenzyme Q10responsive states include, for example, metabolic disorders such asobesity, diabetes, pre-diabetes, Metabolic Syndrome, satiety, andendocrine abnormalities. Coenzyme Q10 responsive states further includeother metabolic disorders as described herein.

In some embodiments, the compounds of the present invention, e.g., theMIMs or epi-shifters described herein, share a common activity withCoenzyme Q10. As used herein, the phrase “share a common activity withCoenzyme Q10” refers to the ability of a compound to exhibit at least aportion of the same or similar activity as Coenzyme Q10. In someembodiments, the compounds of the present invention exhibit 25% or moreof the activity of Coenzyme Q10. In some embodiments, the compounds ofthe present invention exhibit up to and including about 130% of theactivity of Coenzyme Q10. In some embodiments, the compounds of thepresent invention exhibit about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%,107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%,119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, or130% of the activity of Coenzyme Q10. It is to be understood that eachof the values listed in this paragraph may be modified by the term“about.” Additionally, it is to be understood that any range which isdefined by any two values listed in this paragraph is meant to beencompassed by the present invention. For example, in some embodiments,the compounds of the present invention exhibit between about 50% andabout 100% of the activity of Coenzyme Q10. In some embodiments, theactivity shared by Coenzyme Q10 and the compounds of the presentinvention is the ability to induce a shift in cellular metabolism. Incertain embodiments, the activity shared by of CoQ10 and the compoundsof the present invention is measured by OCR (Oxygen Consumption Rate)and/or ECAR (ExtraCellular Acidification Rate).

The present invention provides methods for treating, alleviatingsymptoms of, inhibiting progression of, or preventing a CoQ10 responsivedisorder in a mammal, the method comprising administering to the mammalin need thereof a therapeutically effective amount of pharmaceuticalcomposition comprising at least one environmental influencer(env-influencer), wherein the environmental influencer selectivelyelicits, in a disease cell of the mammal, a cellular metabolic energyshift towards levels of glycolysis and mitochondrial oxidativephosphorylation observed in a normal cell of the mammal under normalphysiological conditions.

The present invention further provides methods for treating, alleviatingsymptoms of, inhibiting progression of, or preventing a metabolicdisorder in a mammal, the method comprising administering to the mammalin need thereof a therapeutically effective amount of a pharmaceuticalcomposition comprising at least one environmental influencer(env-influencer), wherein the environmental influencer selectivelyelicits, in a disease cell of the mammal, a cellular metabolic energyshift towards normalized mitochondrial oxidative phosphorylation.

The present invention further provides methods for selectivelyaugmenting mitochondrial oxidative phosphorylation, in a disease cell ofa mammal in need of treatment for a metabolic disorder, the methodcomprising administering to said mammal a therapeutically effectiveamount of a pharmaceutical composition comprising at least oneenv-influencer, thereby selectively augmenting mitochondrial oxidativephosphorylation in said disease cell of the mammal.

The present invention further provides methods of treating or preventinga metabolic disorder in a human, comprising administering anenvironmental influencer to the human in an amount sufficient to treator prevent the metabolic disorder, thereby treating or preventing themetabolic disorder.

The present invention further provides methods of treating or preventingan metabolic disorder in a human, comprising selecting a human subjectsuffering from an metabolic disorder, and administering to said human atherapeutically effective amount of an env-influencer capable ofblocking anaerobic use of glucose and augmenting mitochondrial oxidativephosphorylation, thereby treating or preventing the metabolic disorder.

The present invention still further provides a method for treating orpreventing a metabolic disorder in a human, comprising administering anenvironmental influencer (env-influencer) to the human in an amountsufficient to treat or prevent the metabolic disorder, wherein theenvironmental influencer (env-influencer) is administered such that itis maintained in its oxidized form during treatment, thereby treating orpreventing the metabolic disorder.

By “a metabolic disorder” is meant any pathological condition resultingfrom an alteration in a patient's metabolism. Such disorders includethose associated with aberrant whole-body glucose, lipid and/or proteinmetabolism and pathological consequences arising therefrom. Metabolicdisorders include those resulting from an alteration in glucosehomeostasis resulting, for example, in hyperglycemia. According to thisinvention, an alteration in glucose levels is typically an increase inglucose levels by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or even 100% relative to such levels in a healthy individual.Metabolic disorders can detrimentally affect cellular functions such ascellular proliferation, growth, differentiation, or migration, cellularregulation of homeostasis, inter- or intra-cellular communication;tissue function, such as liver function, muscle function, or adipocytefunction; systemic responses in an organism, such as hormonal responses(e.g., insulin response). Metabolic disorders include, but are notlimited to, obesity, diabetes (also referred to herein as diabetesmellitus) (e.g., diabetes type I, diabetes type II, MODY, andgestational diabetes), pre-diabetes, Metabolic Syndrome, satiety, andendocrine abnormalities, e.g., of aging. Further examples of metabolicdisorders include, but are not limited to, hyperphagia, hypophagia,triglyceride storage disease, Bardet-Biedl syndrome, Lawrence-Moonsyndrome, Prader-Labhart-Willi syndrome, Kearns-Sayre syndrome,anorexia, medium chain acyl-CoA dehydrogenase deficiency, and cachexia.In some embodiments the metabolic disorder is a Coenzyme Q10 responsivestate.

By “treating, reducing, or preventing a metabolic disorder” is meantameliorating such a condition before or after it has occurred. Ascompared with an equivalent untreated control, such reduction or degreeof prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or100% as measured by any standard technique. Diabetes mellitus is aheterogeneous group of metabolic diseases which lead to chronicelevation of glucose in the blood (hyperglycemia). Diabetes ischaracterized by pancreatic islet destruction or dysfunction leading toloss of glucose regulation. The two major types of diabetes mellitus areType I, also known as “insulin-dependent diabetes” (“IDDM”) or“juvenile-onset diabetes”, and Type II, also known as “non-insulindependent” (“NIDDM”) or “maturity-onset diabetes”.

“Type I diabetes” refers to a condition that results from anautoimmune-mediated destruction of pancreatic .beta. cells withconsequent loss of insulin production, which results in hyperglycemia.Type I diabetics require insulin replacement therapy to ensure survival.While medications such as injectable insulin and oral hypoglycemicsallow diabetics to live longer, diabetes remains the third major killer,after heart disease and cancer. However, these medications do notcontrol blood sugar levels well enough to prevent swinging between highand low blood sugar levels, with resulting damage to the kidneys, eyes,and blood vessels. Data from the Diabetes Control and ComplicationsTrial (DCCT) show that intensive control of blood glucose significantlydelays complications of diabetes, such as retinopathy, nephropathy, andneuropathy, compared with conventional therapy consisting of one or twoinsulin injections per day. Intensive therapy in the DCCT includedmultiple injection of insulin three or more times per day or continuoussubcutaneous insulin infusion (CSII) by external pump. Insulin pumps areone of a variety of alternative approaches to subcutaneous multipledaily injections (MDI) for approximating physiological replacement ofinsulin.

“Type 2 diabetes” refers to the condition in which a patient has afasting blood glucose or serum glucose concentration greater than 125mg/dl (6.94 mmol/L). Type II diabetes is characterized by hyperglycemiain the presence of higher-than-normal levels of plasma insulin(hyperinsulinemia) and represents over 90% of all cases and occurs mostoften in overweight adults over 40 years of age. Progression of Type IIdiabetes is associated with increasing concentrations of blood glucose,coupled with a relative decrease in the rate of glucose-induced insulinsecretion. In Type II diabetes, tissue processes which controlcarbohydrate metabolism are believed to have decreased sensitivity toinsulin and therefore occur not from a lack of insulin production, but adecreased sensitivity to increased glucose levels in the blood and aninability to respond by producing insulin. Alternatively, diabetes mayresult from various defects in the molecular machinery that mediate theaction of insulin on its target cells, such as a lack of insulinreceptors on their cell surfaces. Treatment of Type II diabetestherefore frequently does not require administration of insulin but maybe based on diet and lifestyle changes, augmented by therapy with oralhypoglycemic agents such as, for example, sulfonylurea.

“Pre-diabetes” refers to a condition where a patient is pre-disposed tothe development of type 2 diabetes. Pre-diabetes extends the definitionof impaired glucose tolerance to include individuals with a fastingblood glucose within the high normal range.gtoreq.100 mg/dL (Meigs etal., Diabetes 2003 52:1475-1484) and fasting hyperinsulinemia (elevatedplasma insulin concentration).

“Obesity” refers to the condition where a patient has a BMI equal to orgreater than 30 kg/m.sup.2. “Visceral obesity” refers to a waist to hipration of 1.0 in male patients and 0.8 in female patients. In anotheraspect, visceral obesity defines the risk for insulin resistance and thedevelopment of pre-diabetes.

“Overweight” refers to a patient with a BMI greater than or 25kg/m.sup.2 and less than 30 kg/m.sup.2. “Weight gain” refers to theincrease in body weight in relationship to behavioral habits oraddictions, e.g., overeating or gluttony, smoking cessation, or inrelationship to biological (life) changes, e.g., weight gain associatedwith aging in men and menopause in women or weight gain after pregnancy.

“Metabolic Syndrome” (MS), also referred to as Syndrome X, refers to ametabolic disorder that affects other pathways and systems in the body.Originally, Metabolic Syndrome was defined as a cluster of metabolicdisorders (including obesity, insulin resistance, hypertension, anddyslipidemia primarily hypertriglyceridemia), that synergize topotentiate cardiovascular disease. More recently (2001), the U.S.National Cholesterol Education Program (NCEP) has classified “MetabolicSyndrome” as meeting any three out of the following five criteria:fasting glucose level of at least 110 mg/dl, plasma triglyceride levelof at least 150 mg/dl (hypertriglycerdemia), HDL cholesterol below 40mg/dl in men or below 50 mg/dl in women, blood pressure at least 130/85mm Hg (hypertension), and central obesity, with central obesity beingdefined as abdominal waist circumference greater than 40 inches for menand greater than 35 inches for women. Presently, there are three otherinternationally recognized definitions for Metabolic Syndrome asfollows: 1) World Health Organization 2) American HeartAssociation/National Heart, Lung and blood Institute (AHA/NHLBI) and 3)International Diabetes Federation (IDF). The definitions of MetabolicSyndrome by the WHO, AHA/NHLBI and IDF are very similar to thedefinition of the NECP and all use the same metabolic parameters todefine the syndrome, but the WHO also includes assessment of insulinfasting insulin levels (Moebus S et al, Cardiovascular Diabetology, 6:1-10, 2007; Athyros V G et al, Int. J. Cardiology, 117: 204-210, 2007).Yet subtle differences in the thresholds for these metabolic parametersrequired to be classified as having the syndrome among these differentdefinitions can result in different classification of a particularsubject as having or not having the syndrome according to thesedifferent definitions. Also, the prevalence of cardiovascular disease(CVD) with MS varies by the definition used. (Moebus S et al,Cardiovascular Diabetology, 6: 1-10, 2007; Athyros V G et al, Int. J.Cardiology, 117: 204-210, 2007). The American Diabetes Associationestimates that 1 in every 5 overweight people suffer from MetabolicSyndrome.

In other aspects, the metabolic syndrome is described by acceptedsynonyms, which includes, but is not limited to, syndrome X, insulinresistance syndrome, insulin-resistant hypertension, the metabolichypertensive syndrome, dysmetabolic syndrome. Components of themetabolic syndrome include, but are not limited to, glucose intolerance,impaired glucose tolerance, impaired fasting serum glucose, impairedfasting blood glucose, hyperinsulinemia, pre-diabetes, obesity, visceralobesity, hypertriglyceridemia, elevated serum concentrations of freefatty acids, elevated serum concentrations of C-reactive protein,elevated serum concentrations of lipoprotein(a), elevated serumconcentrations of homocysteine, elevated serum concentrations of small,dense low-density lipoprotein (LDL)-cholesterol, elevated serumconcentrations of lipoprotein-associated phospholipase (A2), reducedserum concentrations of high density lipoprotein (HDL)-cholesterol,reduced serum concentrations of HDL(2b)-cholesterol, reduced serumconcentrations of adiponectin, and albuminuria (see: Pershadsingh HA.Peroxisome proliferator-activated receptor-gamma: therapeutic target fordiseases beyond diabetes: quo vadis? Expert Opin Investig Drugs. (2004)13:215-28, and references cited therein).

The “key elements” of the foregoing metabolic disorders include but arenot limited to, impaired fasting glucose or impaired glucose tolerance,increased waist circumference, increased visceral fat content, increasedfasting plasma glucose, increased fasting plasma triglycerides,decreased fasting high density lipoprotein level, increased bloodpressure, insulin resistance, hyperinsulinemia, cardiovascular disease(or components thereof such as arteriosclerosis, coronary arterydisease, peripheral vascular disease, or cerebrovascular disease),congestive heart failure, elevated plasma norepinephrine, elevatedcardiovascular-related inflammatory factors, elevated plasma factorspotentiating vascular endothelial dysfunction, hyperlipoproteinemia,arteriosclerosis or atherosclerosis, hyperphagia, hyperglycemia,hyperlipidemia, and hypertension or high blood pressure, increasedplasma postprandial triglyceride or free fatty acid levels, increasedcellular oxidative stress or plasma indicators thereof, increasedcirculating hypercoagulative state, hepatic steatosis, hetapticsteatosis, renal disease including renal failure and renalinsufficiency.

“Insulin resistance” refers to a condition in which circulating insulinlevels in excess of the normal response to a glucose load are requiredto maintain the euglycemic state (Ford et al., JAMA. 2002, 287:356-9).Insulin resistance and the response of a patient with insulin resistanceto therapy, may be quantified by assessing the homeostasis modelassessment to insulin resistance (HOMA-IR) score, a reliable indicatorof insulin resistance (Katsuki et al., Diabetes Care 2001, 24:362-5). Anestimate of insulin resistance by the homeostasis assessment model(HOMA)-IR score may be calculated by a formula disclosed in Galvin etal., Diabet Med 1992, 9:921-8 where HOMA-IR=[fasting serum insulin(.mu.U/mL)].times.[fasting plasma glucose (mmol/L)/22.5].

“Hyperinsulinemia” is defined as the condition in which a subject withinsulin resistance, with or without euglycemia, in which the fasting orpostprandial serum or plasma insulin concentration is elevated abovethat of normal, lean individuals without insulin resistance, having awaist-to-hip ration<1.0 (for men) or <0.8 (for women).

The term “impaired glucose tolerance” (IGT) is used to describe a personwho, when given a glucose tolerance test, has a blood glucose level thatfalls between normal and hyperglycemic. Such a person is at a higherrisk of developing diabetes although they are not considered to havediabetes. For example, impaired glucose tolerance refers to a conditionin which a patient has a fasting blood glucose concentration or fastingserum glucose concentration greater than 110 mg/dl and less than 126mg/dl (7.00 mmol/L), or a 2 hour postprandial blood glucose or serumglucose concentration greater than 140 mg/dl (7.78 mmol/L) and less than200 mg/dl (11.11 mmol/L).

The condition of “hyperglycemia” (high blood sugar) is a condition inwhich the blood glucose level is too high. Typically, hyperglycemiaoccurs when the blood glucose level rises above 180 mg/dl. Symptoms ofhyperglycemia include frequent urination, excessive thirst and, over alonger time span, weight loss.

The condition of “hypoglycemia” (low blood sugar) is a condition inwhich the blood glucose level is too low. Typically, hypoglycemia occurswhen the blood glucose level falls below 70 mg/dl. Symptoms ofhypoglycemia include moodiness, numbness of the extremities (especiallyin the hands and arms), confusion, shakiness or dizziness. Since thiscondition arises when there is an excess of insulin over the amount ofavailable glucose it is sometimes referred to as an insulin reaction.

(i) Diagnosis of Metabolic Disorders

The methods and compositions of the present invention are useful fortreating any patient that has been diagnosed with or is at risk ofhaving a metabolic disorder, such as diabetes. A patient in whom thedevelopment of a metabolic disorder (e.g., diabetes or obesity) is beingprevented may or may not have received such a diagnosis. One in the artwill understand that patients of the invention may have been subjectedto standard tests or may have been identified, without examination, asone at high risk due to the presence of one or more risk factors.

Diagnosis of metabolic disorders may be performed using any standardmethod known in the art, such as those described herein. Methods fordiagnosing diabetes are described, for example, in U.S. Pat. No.6,537,806, hereby incorporated by reference. Diabetes may be diagnosedand monitored using, for example, urine tests (urinalysis) that measureglucose and ketone levels (products of the breakdown of fat); tests thatmeasure the levels of glucose in blood; glucose tolerance tests; andassays that detect molecular markers characteristic of a metabolicdisorder in a biological sample (e.g., blood, serum, or urine) collectedfrom the mammal (e.g., measurements of Hemoglobin Alc (HbAlc) levels inthe case of diabetes).

A patient who is being treated for a metabolic disorder is one who amedical practitioner has diagnosed as having such a condition. Diagnosismay be performed by any suitable means, such as those described herein.A patient in whom the development of diabetes or obesity is beingprevented may or may not have received such a diagnosis. One in the artwill understand that patients of the invention may have been subjectedto standard tests or may have been identified, without examination, asone at high risk due to the presence of one or more risk factors, suchas family history, obesity, particular ethnicity (e.g., AfricanAmericans and Hispanic Americans), gestational diabetes or delivering ababy that weighs more than nine pounds, hypertension, having apathological condition predisposing to obesity or diabetes, high bloodlevels of triglycerides, high blood levels of cholesterol, presence ofmolecular markers (e.g., presence of autoantibodies), and age (over 45years of age). An individual is considered obese when their weight is20% (25% in women) or more over the maximum weight desirable for theirheight. An adult who is more than 100 pounds overweight, is consideredto be morbidly obese. Obesity is also defined as a body mass index (BMI)over 30 kg/m.sup.2.

Patients may be diagnosed as being at risk or as having diabetes if arandom plasma glucose test (taken at any time of the day) indicates avalue of 200 mg/dL or more, if a fasting plasma glucose test indicates avalue of 126 mg/dL or more (after 8 hours), or if an oral glucosetolerance test (OGTT) indicates a plasma glucose value of 200 mg/dL ormore in a blood sample taken two hours after a person has consumed adrink containing 75 grams of glucose dissolved in water. The OGTTmeasures plasma glucose at timed intervals over a 3-hour period.Desirably, the level of plasma glucose in a diabetic patient that hasbeen treated according to the invention ranges between 160 to 60 mg/dL,between 150 to 70 mg/dL, between 140 to 70 mg/dL, between 135 to 80mg/dL, and preferably between 120 to 80 mg/dL.

Optionally, a hemoglobin Alc (HbAlc) test, which assesses the averageblood glucose levels during the previous two and three months, may beemployed. A person without diabetes typically has an HbAlc value thatranges between 4% and 6%. For every 1% increase in HbAlc, blood glucoselevels increases by approximately 30 mg/dL and the risk of complicationsincreases. Preferably, the HbAlc value of a patient being treatedaccording to the present invention is reduced to less than 9%, less than7%, less than 6%, and most preferably to around 5%. Thus, the HbAlclevels of the patient being treated are preferably lowered by 10%, 20%,30%, 40%, 50%, or more relative to such levels prior to treatment.

Gestational diabetes is typically diagnosed based on plasma glucosevalues measured during the OGTT. Since glucose levels are normally lowerduring pregnancy, the threshold values for the diagnosis of diabetes inpregnancy are lower than in the same person prior to pregnancy. If awoman has two plasma glucose readings that meet or exceed any of thefollowing numbers, she has gestational diabetes: a fasting plasmaglucose level of 95 mg/dL, a 1-hour level of 180 mg/dL, a 2-hour levelof 155 mg/dL, or a 3-hour level of 140 mg/dL.

Ketone testing may also be employed to diagnose type I diabetes. Becauseketones build up in the blood when there is not enough insulin, theyeventually accumulate in the urine. High levels of blood ketones mayresult in a serious condition called ketoacidosis.

According to the guidelines of the American Diabetes Association, to bediagnosed with Type 2 diabetes, an individual must have a fasting plasmaglucose level greater than or equal to 126 mg/dl or a 2-hour oralglucose tolerance test (OGTT) plasma glucose value of greater than orequal to 200 mg/dl (Diabetes Care, 26:S5-S20, 2003).

A related condition called pre-diabetes is defined as having a fastingglucose level of greater than 100 mg/dl but less than 126 mg/dl or a2-hour OGTT plasma glucose level of greater than 140 mg/dl but less than200 mg/dl. Mounting evidence suggests that the pre-diabetes conditionmay be a risk factor for developing cardiovascular disease (DiabetesCare 26:2910-2914, 2003). Prediabetes, also referred to as impairedglucose tolerance or impaired fasting glucose is a major risk factor forthe development of type 2 diabetes mellitus, cardiovascular disease andmortality. Much focus has been given to developing therapeuticinterventions that prevent the development of type 2 diabetes byeffectively treating prediabetes (Pharmacotherapy, 24:362-71, 2004).

Obesity (commonly defined as a Body Mass Index of approximately >30kg/m.sup.2) is often associated with a variety of pathologic conditionssuch as hyperinsulinemia, insulin resistance, diabetes, hypertension,and dyslipidemia. Each of these conditions contributes to the risk ofcardiovascular disease.

Along with insulin resistance, hypertension, and dyslipidemia, obesityis considered to be a component of the Metabolic Syndrome (also known asSyndrome X) which together synergize to potentiate cardiovasculardisease. More recently, the U.S. National Cholesterol Education Programhas classified Metabolic Syndrome as meeting three out of the followingfive criteria: fasting glucose level of at least 110 mg/dl, plasmatriglyceride level of at least 150 mg/dl (hypertriglycerdemia), HDLcholesterol below 40 mg/dl in men or below 50 mg/dl in women, bloodpressure at least 130/85 mm Hg (hypertension), and central obesity, withcentral obesity being defined as abdominal waist circumference greaterthan 40 inches for men and greater than 35 inches for women.

(ii) Assessing Treatment Efficacy of a Metabolic Disorder

The skilled artisan will recognize that the use of any of the abovetests or any other tests known in the art may be used to monitor theefficacy of the therapeutic treatments of the invention. Since themeasurements of hemoglobin Alc (HbAlc) levels is an indication ofaverage blood glucose during the previous two to three months, this testmay be used to monitor a patient's response to diabetes treatment.

The therapeutic methods of the invention are effective in reducingglucose levels or lipid levels in a patient. By “reducing glucoselevels” is meant reducing the level of glucose by at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative to an untreatedcontrol. Desirably, glucose levels are reduced to normoglycemic levels,i.e., between 150 to 60 mg/dL, between 140 to 70 mg/dL, between 130 to70 mg/dL, between 125 to 80 mg/dL, and preferably between 120 to 80mg/dL. Such reduction in glucose levels may be obtained by increasingany one of the biological activities associated with the clearance ofglucose from the blood. Accordingly, an agent having the ability toreduce glucose levels may increase insulin production, secretion, oraction. Insulin action may be increased, for example, by increasingglucose uptake by peripheral tissues and/or by reducing hepatic glucoseproduction. Alternatively, the agent of the invention may reduce theabsorption of carbohydrates from the intestines, alter glucosetransporter activity (e.g., by increasing GLUT4 expression, intrinsicactivity, or translocation), increase the amount of insulin-sensitivetissue (e.g., by increasing muscle cell or adipocyte celldifferentiation), or alter gene transcription in adipocytes or musclecells (e.g., altered secretion of factors from adipocytes expression ofmetabolic pathway genes). Desirably, the agent of the inventionincreases more than one of the activities associated with the clearanceof glucose. By “reducing lipid levels” is meant reducing the level oflipids by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, or 100% relative to an untreated control.

By “alter insulin signaling pathway such that glucose levels arereduced” is meant to alter (by increasing or reducing) any one of theactivities involved in insulin signaling such that the overall result isan increase in the clearance of glucose from plasma. For example, theenv-influencer of the invention alters the insulin signaling pathwaycausing an increase in insulin production, secretion, or action, anincrease in glucose uptake by peripheral tissues, a reduction in hepaticglucose production, or a reduction in the absorption of carbohydratesfrom the intestines.

The ability of an environmental influencer, e.g., epi-shifter, to reduceglucose levels and thereby treat a metabolic disorder may be assessedusing standard assays known in the art. For example, cell-basedscreening assays that identify agents that increase glucose uptake maybe employed. In particular, differentiated adipocytes in cell culturecan be employed to assess the ability of the epi-shifter to increaseglucose uptake upon insulin stimulation, as detected by radiolabeledglucose. In another exemplary assay, human myoblasts obtained by theconditional immortalization of cells derived from a non-diabetic subjectcan be used to screen the effect of agents on glycogen synthesis, usinginsulin as a positive control. Prior to treatment, cells areserum-starved, and are then incubated either with the epi-shifter orcontrol for a period of two hours in serum-free media containingradiolabeled glucose, after which, glycogen synthesis is measured.Exemplary assays are further described in the Examples.

V. Therapeutic Targets for Metabolic Disorders

The present invention provides methods for identifying therapeutictargets for metabolic disorders. The invention further providestherapeutic targets identified by such methods. The identification of atherapeutic target involves, generally, the exogenous application of anEnv-influencer or candidate Env-influencer to a cell or panel of celllines, and the subsequent evaluation of changes induced to a treatedcell as compared to a control, untreated cell. Induced cellular changeswhich are monitored include, but are not limited to, changes to themorphology, physiology or composition, e.g., RNA, protein, lipid ormetabolite levels, of the cell. Induced cellular changes as a result oftreatment by a candidate Env-influencer can be monitored by using any ofthe assays described herein. For example, changes in gene expression atthe mRNA level can be evaluated by real-time PCR arrays, while changesin gene expression at the protein level can be monitored by usingantibody microarrays and 2-D gel electrophoresis. Genes identified asbeing modulated by the candidate Env-influencer (e.g., at the mRNAand/or protein level) are then evaluated from a Systems Biologyperspective using pathway analysis (Ingenuity IPA software) and by areview of the known literature. Genes identified as potentialtherapeutic targets are next submitted to confirmatory assays such asWestern blot analysis, siRNA knock-down, or recombinant proteinproduction and characterization methods. Screening assays can then beused to identify modulators of the targets. Modulators of thetherapeutic targets are useful as novel therapeutic agents for metabolicdisorders. Modulators of therapeutic targets can be routinely identifiedusing screening assays described in detail herein, or by using routinemethodologies known to the skilled artisan.

Genes identified herein as being modulated (e.g., upmodulated ordownmodulated, at either the mRNA or protein level) by theMIM/Epi-shifter, CoQ10, are drug targets of the invention. Drug targetsof the invention include, but are not limited to, the genes subsequentlylisted in Tables 2-4 & 6-28 & 63-68 herein. Based on the results ofexperiments described by Applicants herein, the key proteins modulatedby Q10 are associated with or can be classified into different pathwaysor groups of molecules, including transcription factors, apoptoticresponse, pentose phosphate pathway, biosynthetic pathway, oxidativestress (pro-oxidant), membrane alterations, and oxidativephosphorylation metabolism. The key proteins modulated by CoQ10, basedon the results provided herein, are summarized as follows. A key proteinmodulated by CoQ10 and which is a transcription factor is HNF4alpha. Keyproteins that are modulated by CoQ10 and associated with the apoptoticresponse include Bcl-xl, Bcl-xl, Bcl-xS, BNIP-2, Bcl-2, Birc6, Bcl-2-L11(Bim), XIAP, BRAF, Bax, c-Jun, Bmf, PUMA, and cMyc. A key protein thatis modulated by CoQ10 and associated with the pentose phosphate pathwayis transaldolase 1. Key proteins that are modulated by CoQ10 andassociated with a biosynthetic pathway include COQ1, COQ3, COQ6,prenyltransferase and 4-hydroxybenzoate. Key proteins that are modulatedby CoQ10 and associated with oxidative stress (pro-oxidant) includeNeutrophil cytosolic factor 2, nitric oxide synthase 2A and superoxidedismutase 2 (mitochondrial). Key proteins that are modulated by CoQ10and associated with oxidative phosphorylation metabolism includeCytochrome c, complex I, complex II, complex III and complex IV. Furtherkey proteins that are directly or indirectly modulated by CoQ10 includeFoxo 3a, DJ-1, IDH-1, Cpt1C and Cam Kinase II.

Accordingly, in one embodiment of the invention, a drug target mayinclude HNF4-alpha, Bcl-x1, Bcl-xS, BNIP-2, Bcl-2, Birc6, Bcl-2-L11(Bim), XIAP, BRAF, Bax, c-Jun, Bmf, PUMA, cMyc, transaldolase 1, COQ1,COQ3, COQ6, prenyltransferase, 4-hydrobenzoate, neutrophil cytosolicfactor 2, nitric oxide synthase 2A, superoxide dismutase 2, VDAC, Baxchannel, ANT, Cytochrome c, complex 1, complex II, complex III, complexIV, Foxo 3a, DJ-1, IDH-1, Cpt1C and Cam Kinase II. In a preferredembodiment, a drug target may include HNF4A, Transaldolase, NM23 andBSCv. In one embodiment, the drug target is TNF4A. In one embodiment,the drug target is transaldolase. In one embodiment, the drug target isNM23. In one embodiment, the drug target is BSCv. Screening assaysuseful for identifying modulators of identified drug targets aredescribed below.

VI. Screening Assays

The invention also provides methods (also referred to herein as“screening assays”) for identifying modulators, i.e., candidate or testcompounds or agents (e.g., proteins, peptides, peptidomimetics,peptoids, small molecules or other drugs), which modulate the expressionand/or activity of an identified therapeutic target of the invention.Such assays typically comprise a reaction between a therapeutic targetof the invention and one or more assay components. The other componentsmay be either the test compound itself, or a combination of testcompounds and a natural binding partner of a marker of the invention.Compounds identified via assays such as those described herein may beuseful, for example, for treating or preventing a metabolic disorder.

The test compounds used in the screening assays of the present inventionmay be obtained from any available source, including systematiclibraries of natural and/or synthetic compounds. Test compounds may alsobe obtained by any of the numerous approaches in combinatorial librarymethods known in the art, including: biological libraries; peptoidlibraries (libraries of molecules having the functionalities ofpeptides, but with a novel, non-peptide backbone which are resistant toenzymatic degradation but which nevertheless remain bioactive; see,e.g., Zuckermann et al., 1994, J. Med. Chem. 37:2678-85); spatiallyaddressable parallel solid phase or solution phase libraries; syntheticlibrary methods requiring deconvolution; the ‘one-bead one-compound’library method; and synthetic library methods using affinitychromatography selection. The biological library and peptoid libraryapproaches are limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam, 1997, Anticancer Drug Des.12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten,1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria and/orspores, (Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al, 1992,Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith, 1990,Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al,1990, Proc. Natl. Acad. Sci. 87:6378-6382; Felici, 1991, J. Mol. Biol.222:301-310; Ladner, supra.).

The screening methods of the invention comprise contacting a cell with atest compound and determining the ability of the test compound tomodulate the expression and/or activity of a therapeutic target of theinvention in the cell. The expression and/or activity of a therapeutictarget of the invention can be determined as described herein. Theexpression and/or activity of a therapeutic target of the invention canalso be determined by using routine methods known to the skilledartisan. In one embodiment, a compound is selected based on its abilityto increase expression and/or activity of a therapeutic target of theinvention. In one embodiment, a compound is selected based on itsability increase expression and/or activity of a therapeutic targetselected from the protein listed in Tables 2-4 & 6-28 & 63-68, whereinthe therapeutic target is upmodulated by CoQ10 (e.g., exhibits apositive-fold change). In one embodiment, a compound is selected basedon its ability to decrese expression and/or activity of a therapeutictarget of the invention. In one embodiment, a compound is selected basedon its ability to decrease expression and/or activity of a therapeutictarget selected from the proteins listed in Tables 2-4 & 6-28 & 63-68,wherein the therapeutic target is downmodulated by CoQ10 (e.g., exhibitsa negative-fold change).

In another embodiment, the invention provides assays for screeningcandidate or test compounds which are substrates of a therapeutic targetof the invention or biologically active portions thereof. In yet anotherembodiment, the invention provides assays for screening candidate ortest compounds which bind to a therapeutic target of the invention orbiologically active portions thereof. Determining the ability of thetest compound to directly bind to a therapeutic target can beaccomplished, for example, by coupling the compound with a radioisotopeor enzymatic label such that binding of the compound to the drug targetcan be determined by detecting the labeled marker compound in a complex.For example, compounds (e.g., marker substrates) can be labeled with¹³¹I, ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and theradioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, assay components can beenzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent capable of modulatingthe expression and/or activity of a marker of the invention identifiedas described herein can be used in an animal model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal model to determine the mechanism of action of such an agent.Furthermore, this invention pertains to uses of novel agents identifiedby the above-described screening assays for treatment as describedabove.

VII. Pharmaceutical Compositions and Pharmaceutical Administration

The environmental influencers of the invention can be incorporated intopharmaceutical compositions suitable for administration to a subject.Typically, the pharmaceutical composition comprises an environmentalinfluencer of the invention and a pharmaceutically acceptable carrier.As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Examples of pharmaceutically acceptablecarriers include one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable carriers may further include minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of theenvironmental influencer.

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, creams, lotions, ointments orpasts, drops suitable for administration to the eye, ear, or nose,liposomes and suppositories. The preferred form depends on the intendedmode of administration and therapeutic application.

The environmental influencers of the present invention can beadministered by a variety of methods known in the art. For manytherapeutic applications, the preferred route/mode of administration issubcutaneous injection, intravenous injection or infusion. As will beappreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results. In certainembodiments, the active compound may be prepared with a carrier thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978. In one embodiment, the mode ofadministration is parenteral (e.g., intravenous, subcutaneous,intraperitoneal, intramuscular). In one embodiment, the environmentalinfluencer is administered by intravenous infusion or injection. Inanother embodiment, the environmental influencer is administered byintramuscular or subcutaneous injection. In a preferred embodiment, theenvironmental influencer is administered topically.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,environmental influencer) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile,lyophilized powders for the preparation of sterile injectable solutions,the preferred methods of preparation are vacuum drying and spray-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Techniques and formulations generally may be found in Remmington'sPharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemicadministration, injection is preferred, including intramuscular,intravenous, intraperitoneal, and subcutaneous. For injection, thecompounds of the invention can be formulated in liquid solutions,preferably in physiologically compatible buffers such as Hank's solutionor Ringer's solution. In addition, the compounds may be formulated insolid form and redissolved or suspended immediately prior to use.Lyophilized forms are also included.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound. For buccal administration thecompositions may take the form of tablets or lozenges formulated inconventional manner. For administration by inhalation, the compounds foruse according to the present invention are conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration bile salts and fusidic acidderivatives in addition, detergents may be used to facilitatepermeation. Transmucosal administration may be through nasal sprays orusing suppositories. For topical administration, the compound(s) of theinvention are formulated into ointments, salves, gels, or creams asgenerally known in the art. A wash solution can be used locally to treatan injury or inflammation to accelerate healing.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

For therapies involving the administration of nucleic acids, thecompound(s) of the invention can be formulated for a variety of modes ofadministration, including systemic and topical or localizedadministration. Techniques and formulations generally may be found inRemmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa.For systemic administration, injection is preferred, includingintramuscular, intravenous, intraperitoneal, intranodal, andsubcutaneous. For injection, the compound(s) of the invention can beformulated in liquid solutions, preferably in physiologically compatiblebuffers such as Hank's solution or Ringer's solution. In addition, thecompound(s) may be formulated in solid form and redissolved or suspendedimmediately prior to use. Lyophilized forms are also included.

In one embodiment, the compositions comprising an Environmentalinfluencer are administered topically. It is preferable to present theactive ingredient, i.e. Env-influencer, as a pharmaceutical formulation.The active ingredient may comprise, for topical administration, fromabout 0.001% to about 20% w/w, by weight of the formulation in the finalproduct, although it may comprise as much as 30% w/w, preferably fromabout 1% to about 20% w/w of the formulation. The topical formulationsof the present invention, comprise an active ingredient together withone or more acceptable carrier(s) therefor and optionally any othertherapeutic ingredients(s). The carrier(s) should be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipient thereof.

In treating a patient exhibiting a disorder of interest, atherapeutically effective amount of an agent or agents such as these isadministered. A therapeutically effective dose refers to that amount ofthe compound that results in amelioration of symptoms or a prolongationof survival in a patient.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Thedata obtained from these cell culture assays and animal studies can beused in formulating a range of dosage for use in human. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating plasma concentration range that includes theIC₅₀ as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by HPLC.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (Seee.g. Fingl et al., in The Pharmacological Basis of Therapeutics, 1975,Ch. 1 p. 1). It should be noted that the attending physician would knowhow to and when to terminate, interrupt, or adjust administration due totoxicity, or to organ dysfunctions. Conversely, the attending physicianwould also know to adjust treatment to higher levels if the clinicalresponse were not adequate (precluding toxicity). The magnitude of anadministrated dose in the management of the oneogenic disorder ofinterest will vary with the severity of the condition to be treated andto the route of administration. The severity of the condition may, forexample, be evaluated, in part, by standard prognostic evaluationmethods. Further, the dose and perhaps dose frequency, will also varyaccording to the age, body weight, and response of the individualpatient. A program comparable to that discussed above may be used inveterinary medicine.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in Remington'sPharmaceutical Sciences, 18^(th) ed., Mack Publishing Co., Easton, Pa.(1990). Suitable routes may include oral, rectal, transdermal, vaginal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections, just to name afew.

The compositions described above may be administered to a subject in anysuitable formulation. In addition to treatment of a metabolic disorderwith topical formulations of an environmental influencer, e.g., CoQ10,in other aspects of the invention the environmental influencer, e.g.,CoQ10, might be delivered by other methods. For example, theenvironmental influencer, e.g., CoQ10, might be formulated forparenteral delivery, e.g., for subcutaneous, intravenous, intramuscular,or intratumoral injection. Other methods of delivery, for example,liposomal delivery or diffusion from a device impregnated with thecomposition might be used. The compositions may be administered in asingle bolus, multiple injections, or by continuous infusion (forexample, intravenously or by peritoneal dialysis). For parenteraladministration, the compositions are preferably formulated in asterilized pyrogen-free form. Compositions of the invention can also beadministered in vitro to a cell (for example, to induce apoptosis in acancer cell in an in vitro culture) by simply adding the composition tothe fluid in which the cell is contained.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in Remington'sPharmaceutical Sciences, 18.^(th) ed., Mack Publishing Co., Easton, Pa.(1990). Suitable routes may include oral, rectal, transdermal, vaginal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections, just to name afew.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Forsuch transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

Use of pharmaceutically acceptable carriers to formulate the compoundsherein disclosed for the practice of the invention into dosages suitablefor systemic administration is within the scope of the invention. Withproper choice of carrier and suitable manufacturing practice, thecompositions of the present invention, in particular, those formulatedas solutions, may be administered parenterally, such as by intravenousinjection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions. and the like, for oral ingestion bya patient to be treated.

Agents intended to be administered intracellularly may be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents may be encapsulated into liposomes, thenadministered as described above. Liposomes are spherical lipid bilayerswith aqueous interiors. All molecules present in an aqueous solution atthe time of liposome formation are incorporated into the aqueousinterior. The liposomal contents are both protected from the externalmicroenvironment and, because liposomes fuse with cell membranes, areefficiently delivered into the cell cytoplasm. Additionally, due totheir hydrophobicity, small organic molecules may be directlyadministered intracellularly.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein. Inaddition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions. The pharmaceuticalcompositions of the present invention may be manufactured in a mannerthat is itself known, e.g., by means of conventional mixing, dissolving,granulating, dragee-making, levitating, emulsifying, encapsulating,entrapping or lyophilizing processes.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of where treatment is required, such as liniments, lotions,creams, ointments or pastes, and drops suitable for administration tothe eye, ear, or nose. Drops according to the present invention maycomprise sterile aqueous or oily solutions or suspensions and may beprepared by dissolving the active ingredient in a suitable aqueoussolution of a bactericidal and/or fungicidal agent and/or any othersuitable preservative, and preferably including a surface active agent.The resulting solution may then be clarified and sterilized byfiltration and transferred to the container by an aseptic technique.Examples of bactericidal and fungicidal agents suitable for inclusion inthe drops are phenylmercuric nitrate or acetate (0.002%), benzalkoniumchloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solventsfor the preparation of an oily solution include glycerol, dilutedalcohol and propylene glycol.

Lotions according to the present invention include those suitable forapplication to the skin or eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those for the preparation of drops. Lotions orliniments for application to the skin may also include an agent tohasten drying and to cool the skin, such as an alcohol or acetone,and/or a moisturizer such as glycerol or an oil such as castor oil orarachis oil.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient infinely-divided or powdered form, alone or in solution or suspension inan aqueous or non-aqueous fluid, with the aid of suitable machinery,with a greasy or non-greasy basis. The basis may comprise hydrocarbonssuch as hard, soft or liquid paraffin, glycerol, beeswax, a metallicsoap; a mucilage; an oil of natural origin such as almond, corn,arachis, castor or olive oil; wool fat or its derivatives, or a fattyacid such as stearic or oleic acid together with an alcohol such aspropylene glycol or macrogels. The formulation may incorporate anysuitable surface active agent such as an anionic, cationic or non-ionicsurface active such as sorbitan esters or polyoxyethylene derivativesthereof. Suspending agents such as natural gums, cellulose derivativesor inorganic materials such as silicaceous silicas, and otheringredients such as lanolin, may also be included.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxy-methylcellulose, and/orpolyvinyl pyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coating. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

The composition can include a buffer system, if desired. Buffer systemsare chosen to maintain or buffer the pH of compositions within a desiredrange. The term “buffer system” or “buffer” as used herein refers to asolute agent or agents which, when in a water solution, stabilize suchsolution against a major change in pH (or hydrogen ion concentration oractivity) when acids or bases are added thereto. Solute agent or agentswhich are thus responsible for a resistance or change in pH from astarting buffered pH value in the range indicated above are well known.While there are countless suitable buffers, potassium phosphatemonohydrate is a preferred buffer.

The final pH value of the pharmaceutical composition may vary within thephysiological compatible range. Necessarily, the final pH value is onenot irritating to human skin and preferably such that transdermaltransport of the active compound, i.e. CoQ10 is facilitated. Withoutviolating this constraint, the pH may be selected to improve CoQ10compound stability and to adjust consistency when required. In oneembodiment, the preferred pH value is about 3.0 to about 7.4, morepreferably about 3.0 to about 6.5, most preferably from about 3.5 toabout 6.0.

For preferred topical delivery vehicles the remaining component of thecomposition is water, which is necessarily purified, e.g., deionizedwater. Such delivery vehicle compositions contain water in the range ofmore than about 50 to about 95 percent, based on the total weight of thecomposition. The specific amount of water present is not critical,however, being adjustable to obtain the desired viscosity (usually about50 cps to about 10,000 cps) and/or concentration of the othercomponents. The topical delivery vehicle preferably has a viscosity ofat least about 30 centipoises.

Other known transdermal skin penetration enhancers can also be used tofacilitate delivery of CoQ10. Illustrative are sulfoxides such asdimethylsulfoxide (DMSO) and the like; cyclic amides such as1-dodecylazacycloheptane-2-one (Azone™, a registered trademark of NelsonResearch, Inc.) and the like; amides such as N,N-dimethyl acetamide(DMA) N,N-diethyl toluamide, N,N-dimethyl formamide, N,N-dimethyloctamide, N,N-dimethyl decamide, and the like; pyrrolidone derivativessuch as N-methyl-2-pyrrolidone, 2-pyrrolidone,2-pyrrolidone-5-carboxylic acid, N-(2-hydroxyethyl)-2-pyrrolidone orfatty acid esters thereof, 1-lauryl-4-methoxycarbonyl-2-pyrrolidone,N-tallowalkylpyrrolidones, and the like; polyols such as propyleneglycol, ethylene glycol, polyethylene glycol, dipropylene glycol,glycerol, hexanetriol, and the like; linear and branched fatty acidssuch as oleic, linoleic, lauric, valeric, heptanoic, caproic, myristic,isovaleric, neopentanoic, trimethyl hexanoic, isostearic, and the like;alcohols such as ethanol, propanol, butanol, octanol, oleyl, stearyl,linoleyl, and the like; anionic surfactants such as sodium laurate,sodium lauryl sulfate, and the like; cationic surfactants such asbenzalkonium chloride, dodecyltrimethylammonium chloride,cetyltrimethylammonium bromide, and the like; non-ionic surfactants suchas the propoxylated polyoxyethylene ethers, e.g., Poloxamer 231,Poloxamer 182, Poloxamer 184, and the like, the ethoxylated fatty acids,e.g., Tween 20, Myjr 45, and the like, the sorbitan derivatives, e.g.,Tween 40, Tween 60, Tween 80, Span 60, and the like, the ethoxylatedalcohols, e.g., polyoxyethylene (4) lauryl ether (Brij 30),polyoxyethylene (2) oleyl ether (Brij 93), and the like, lecithin andlecithin derivatives, and the like; the terpenes such as D-limonene,α-pinene, β-carene, α-terpineol, carvol, carvone, menthone, limoneneoxide, α-pinene oxide, eucalyptus oil, and the like. Also suitable asskin penetration enhancers are organic acids and esters such assalicyclic acid, methyl salicylate, citric acid, succinic acid, and thelike.

In one embodiment, the present invention provides CoQ10 compositions andmethods of preparing the same. Preferably, the compositions comprise atleast about 1% to about 25% CoQ10 w/w. CoQ10 can be obtained from AsahiKasei N&P (Hokkaido, Japan) as UBIDECARENONE (USP). CoQ10 can also beobtained from Kaneka Q10 as Kaneka Q10 (USP UBIDECARENONE) in powderedform (Pasadena, Tex., USA). CoQ10 used in the methods exemplified hereinhave the following characteristics: residual solvents meet USP 467requirement; water content is less than 0.0%, less than 0.05% or lessthan 0.2%; residue on ignition is 0.0%, less than 0.05%, or less than0.2% less than; heavy metal content is less than 0.002%, or less than0.001%; purity of between 98-100% or 99.9%, or 99.5%. Methods ofpreparing the compositions are provided in the examples section below.

In certain embodiments of the invention, methods are provided fortreating or preventing a metabolic disorder in a human by topicallyadministering Coenzyme Q10 to the human such that treatment orprevention occurs, wherein the human is administered a topical dose ofCoenzyme Q10 in a topical vehicle where Coenzyme Q10 is applied to thetarget tissue in the range of about 0.01 to about 0.5 milligrams ofcoenzyme Q10 per square centimeter of skin. In one embodiment, CoenzymeQ10 is applied to the target tissue in the range of about 0.09 to about0.15 mg CoQ10 per square centimeter of skin. In various embodiments,Coenzyme Q10 is applied to the target tissue in the range of about 0.001to about 5.0, about 0.005 to about 1.0, about 0.005 to about 0.5, about0.01 to about 0.5, about 0.025 to about 0.5, about 0.05 to about 0.4,about 0.05 to about 0.30, about 0.10 to about 0.25, or about 0.10 to0.20 mg CoQ10 per square centimeter of skin. In other embodiments,Coenzyme Q10 is applied to the target tissue at a dose of about 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13,0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25,0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37,0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49or 0.5 mg CoQ10 per square centimeter of skin. In one embodiment,Coenzyme Q10 is applied to the target tissue at a dose of about 0.12 mgCoQ10 per square centimeter of skin It should be understood that rangeshaving any one of these values as the upper or lower limits are alsointended to be part of this invention, e.g., about 0.03 to about 0.12,about 0.05 to about 0.15, about 0.1 to about 0.20, or about 0.32 toabout 0.49 mg CoQ10 per square centimeter of skin.

In another embodiment of the invention, the Coenzyme Q10 is administeredin the form of a CoQ10 cream at a dosage of between 0.5 and 10milligrams of the CoQ10 cream per square centimeter of skin, wherein theCoQ10 cream comprises between 1 and 5% of Coenzyme Q10. In oneembodiment, the CoQ10 cream comprises about 3% of Coenzyme Q10. In otherembodiments, the CoQ10 cream comprises about 1%, 1.5%, 2%, 2.5%, 3%,3.5%, 4%, 4.5% or 5% of Coenzyme Q10. In various embodiments, the CoQ10cream is administered at a dosage of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0,3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10milligrams of CoQ10 cream per square centimeter of skin. It should beunderstood that ranges having any one of these values as the upper orlower limits are also intended to be part of this invention, e.g.,between about 0.5 and about 5.0, about 1.5 and 2.5, or about 2.5 and 5.5mg CoQ10 cream per square centimeter of skin.

In another embodiment, the Coenzyme Q10 is administered in the form of aCoQ10 cream at a dosage of between 3 and 5 milligrams of the CoQ10 creamper square centimeter of skin, wherein the CoQ10 cream comprises between1 and 5% of Coenzyme Q10. In one embodiment, the CoQ10 cream comprisesabout 3% of Coenzyme Q10. In other embodiments, the CoQ10 creamcomprises about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% of CoenzymeQ10. In various embodiments, the CoQ10 cream is administered at a dosageof about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1,4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0 milligrams of CoQ10 creamper square centimeter of skin. It should be understood that rangeshaving any one of these values as the upper or lower limits are alsointended to be part of this invention, e.g., between about 3.0 and about4.0, about 3.3 and 5.3, or about 4.5 and 4.9 mg CoQ10 cream per squarecentimeter of skin.

Certain aspects of the invention provide methods for treating orpreventing a metabolic disorder in a human by topically administeringCoenzyme Q10 to the human such that treatment or prevention occurs,wherein the Coenzyme Q10 is topically applied one or more times per 24hours for six weeks or more.

Certain aspects of the invention provide methods for the preparation ofa Coenzyme Q10 cream 3% which includes the steps of preparing a Phase A,B, C, D and E and combining all the phases such that an oil-in-wateremulsion of 3% CoQ10 cream is formed.

In some embodiments, the Phase A ingredients include Alkyl C₁₂₋₁₅benzoate NF at 4.00% w/w, cetyl alcohol NF at 2.00% w/w, glycerylstearate/PEG-100 at 4.5% w/w and stearyl alcohol NF at 1.50% w/w whilethe Phase B ingredients include diethylene glycol monoethyl ether NF at5.00% w/w, glycerin USP at 2.00% w/w, propylene glycol USP at 1.50% w/w,phenoxyethanol NF at 0.475% w/w, purified water USP at 16.725% w/w andCarbomer Dispersion 2% at 40.00% w/w and the Phace C ingredients includelactic acid USP at 0.50% w/w, sodium lactate solution USP at 2.00% w/w,trolamine NF at 1.30% w/w, and purified water USP at 2.50% w/w.Furthermore in these embodiments the Phase D ingredients includetitanium dioxide USP at 1.00% w/w while the Phase E ingredients includeCoQ10 21% concentrate at 15% w/w.

The term “Trolamine,” as used herein, refers to Trolamine NF,Triethanolamine, TEAlan®, TEAlan 99%, Triethanolamine, 99%,Triethanolamine, NF or Triethanolamine, 99%, NF. These terms may be usedinterchangeably herein.

In certain other embodiments, the Phase A ingredients includecapric/caprylic triglyceride at 4.00% w/w, cetyl alcohol NF at 2.00%w/w, glyceril stearate/PEG-100 at 4.5% and stearyl alcohol NF at 1.5%w/w while the Phase B ingredients include diethylene glycol monoethylether NF at 5.00% w/w, glycerin USP at 2.00% w/w, propylene glycol USPat 1.50% w/w, phenoxyethanol NF at 0.475% w/w, purified water USP at16.725% w/w and Carbomer Dispersion 2% at 40.00% w/w and the Phace Cingredients include lactic acid USP at 0.50% w/w, sodium lactatesolution USP at 2.00% w/w, trolamine NF at 1.30% w/w, and purified waterUSP at 2.50% w/w. Furthermore in these embodiments the Phase Dingredients include titanium dioxide USP at 1.00% w/w while the Phase Eingredients include CoQ10 21% concentrate at 15% w/w.

In certain embodiments of the invention, methods are provided for thepreparation of a Coenzyme Q10 cream 3% which include the steps of (1)adding the Phase A ingredients to a suitable container and heating to70-80 degrees C. in a water bath; (2) adding the Phase B ingredients,excluding the Carbomer Dispersion, to a suitable container and mixing toform a mixed Phase B; (3) placing the Phase E ingredients into asuitable container and melting them at 50-60 degrees C. using a waterbath to form a melted Phase E; (4) adding the Carbomer Dispersion to aMix Tank and heating to 70-80 degrees C. while mixing; (5) adding themixed Phase B to the Mix Tank while maintaining the temperature at 70-80degrees C.; (6) adding the Phase C ingredients to the Mix Tank whilemaintaining the temperature at 70-80 degrees C.; (7) adding the Phase Dingredients to the Mix Tank and then continue mixing and homogenizingthe contents of the Mix Tank; then (8) stopping the homogenization andcooling the contents of the Mix Tank to 50-60 degrees C.; then (9)discontinuing the mixing and adding the melted Phase E to the Mix Tankto form a dispersion; (10) mixing is then resumed until the dispersionis smooth and uniform; then (11) cooling the contents of the Mix Tank to45-50 degrees C.

In some other embodiments of the invention, a pharmaceutical compositioncomprising CoQ10 cream 3% is provided. The cream includes a phase Ahaving C₁₂₋₁₅ alkyl benzoate at 4.00% w/w of the composition, cetylalcohol at 2.00% w/w of the composition, stearyl alcohol at 1.5% w/w,glyceryl stearate and PEG-100 at 4.5% w/w; a phase B having glycerin at2.00% w/w, propylene glycol at 1.5% w/w, ethoxydiglycol at 5.0% w/w,phenoxyethanol at 0.475% w/w, a carbomer dispersion at 40.00% w/w,purified water at 16.725% w/w; a phase C having triethanolamine at1.300% w/w, lactic acid at 0.500% w/w, sodium lactate solution at 2.000%w/w, water at 2.5% w/w; a phase D having titanium dioxide at 1.000% w/w;and a phase E having CoQ10 21% concentrate at 15.000% w/w. In someembodiments the Carbomer Dispersion includes water, phenoxyethanol,propylene glycol and Carbomer 940.

In some other embodiments of the invention, a pharmaceutical compositioncomprising CoQ10 cream 3% is provided. The cream includes a phase Ahaving Capric/Caprylic triglyceride at 4.00% w/w of the composition,cetyl alcohol at 2.00% w/w of the composition, stearyl alcohol at 1.5%w/w, glyceryl stearate and PEG-100 at 4.5% w/w; a phase B havingglycerin at 2.00% w/w, propylene glycol at 1.5% w/w, ethoxydiglycol at5.0% w/w, phenoxyethanol at 0.475% w/w, a carbomer dispersion at 40.00%w/w, purified water at 16.725% w/w; a phase C having triethanolamine at1.300% w/w, lactic acid at 0.500% w/w, sodium lactate solution at 2.000%w/w, water at 2.5% w/w; a phase D having titanium dioxide at 1.000% w/w;and a phase E having CoQ10 21% concentrate at 15.000% w/w. In someembodiments the Carbomer Dispersion includes water, phenoxyethanol,propylene glycol and Carbomer 940.

In some other embodiments of the invention, a pharmaceutical compositioncomprising CoQ10 cream 1.5% is provided. The cream includes a phase Ahaving C₁₂₋₁₅ alkyl benzoate at 5.000% w/w, cetyl alcohol at 2.000% w/w,stearyl alcohol at 1.5% w/w, glyceryl stearate and PEG-100 stearate at4.500% w/w; a phase B having glycerin at 2.000% w/w, propylene at 1.750%w/w, ethoxydiglycol at 5.000% w/w, phenoxyethanol at 0.463% w/w, acarbomer dispersion at 50% w/w, and purified water at 11.377% w/w; aphase C having triethanolamine at 1.3% w/w, lactic acid at 0.400% w/w,sodium lactate solution at 2.000% w/w, and water at 4.210% w/w; a phaseD having titanium dioxide at 1.000% w/w; and a phase E having CoQ10 21%concentrate at 1.500% w/w.

In some other embodiments of the invention, a pharmaceutical compositioncomprising CoQ10 cream 1.5% is provided. The cream includes a phase Ahaving Capric/Caprylic triglyceride at 5.000% w/w, cetyl alcohol at2.000% w/w, stearyl alcohol at 1.5% w/w, glyceryl stearate and PEG-100stearate at 4.500% w/w; a phase B having glycerin at 2.000% w/w,propylene at 1.750% w/w, ethoxydiglycol at 5.000% w/w, phenoxyethanol at0.463% w/w, a carbomer dispersion at 50% w/w, and purified water at11.377% w/w; a phase C having triethanolamine at 1.3% w/w, lactic acidat 0.400% w/w, sodium lactate solution at 2.000% w/w, and water at4.210% w/w; a phase D having titanium dioxide at 1.000% w/w; and a phaseE having CoQ10 21% concentrate at 1.500% w/w. In some embodiments theCarbomer Dispersion includes water, phenoxyethanol and propylene glycol.

1. Combination Therapies

In certain embodiments, an environmental influencer of the inventionand/or pharmaceutical compositions thereof can be used in combinationtherapy with at least one other therapeutic agent, which may be adifferent environmental influencer and/or pharmaceutical compositionsthereof. The environmental influencer and/or pharmaceutical compositionthereof and the other therapeutic agent can act additively or, morepreferably, synergistically. In one embodiment, an environmentalinfluencer and/or a pharmaceutical composition thereof is administeredconcurrently with the administration of another therapeutic agent. Inanother embodiment, a compound and/or pharmaceutical composition thereofis administered prior or subsequent to administration of anothertherapeutic agent.

Examples of other therapeutic agents which can be used with anenvironmental influencer of the invention include, but are not limitedto, diabetes mellitus-treating agents, diabetic complication-treatingagents, antihyperlipemic agents, hypotensive or antihypertensive agents,anti-obesity agents, diuretics, chemotherapeutic agents,immunotherapeutic agents immunosuppressive agents, and the like.

Examples of agents for treating diabetes mellitus include insulinformulations (e.g., animal insulin formulations extracted from apancreas of a cattle or a swine; a human insulin formulation synthesizedby a gene engineering technology using microorganisms or methods),insulin sensitivity enhancing agents, pharmaceutically acceptable salts,hydrates, or solvates thereof (e.g., pioglitazone, troglitazone,rosiglitazone, netoglitazone, balaglitazone, rivoglitazone,tesaglitazar, farglitazar, CLX-0921, R-483, NIP-221, NIP-223, DRF-2189,GW-7282TAK-559, T-131, RG-12525, LY-510929, LY-519818, BMS-298585,DRF-2725, GW-1536, G1-262570, KRP-297, TZD18 (Merck), DRF-2655, and thelike), alpha-glycosidase inhibitors (e.g., voglibose, acarbose,miglitol, emiglitate and the like), biguanides (e.g., phenformin,metformin, buformin and the like) or sulfonylureas (e.g., tolbutamide,glibenclamide, gliclazide, chlorpropamide, tolazamide, acetohexamide,glyclopyramide, glimepiride and the like) as well as other insulinsecretion-promoting agents (e.g., repaglinide, senaglinide, nateglinide,mitiglinide, GLP-1 and the like), amyrin agonist (e.g., pramlintide andthe like), phosphotyrosinphosphatase inhibitor (e.g., vanadic acid andthe like) and the like.

Examples of agents for treating diabetic complications include, but arenot limited to, aldose reductase inhibitors (e.g., tolrestat,epalrestat, zenarestat, zopolrestat, minalrestat, fidareatat, SK-860,CT-112 and the like), neurotrophic factors (e.g., NGF, NT-3, BDNF andthe like), PKC inhibitors (e.g., LY-333531 and the like), advancedglycation end-product (AGE) inhibitors (e.g., ALT946, pimagedine,pyradoxamine, phenacylthiazolium bromide (ALT766) and the like), activeoxygen quenching agents (e.g., thioctic acid or derivative thereof, abioflavonoid including flavones, isoflavones, flavonones, procyanidins,anthocyanidins, pycnogenol, lutein, lycopene, vitamins E, coenzymes Q,and the like), cerebrovascular dilating agents (e.g., tiapride,mexiletene and the like).

Antihyperlipemic agents include, for example, statin-based compoundswhich are cholesterol synthesis inhibitors (e.g., pravastatin,simvastatin, lovastatin, atorvastatin, fluvastatin, rosuvastatin and thelike), squalene synthetase inhibitors or fibrate compounds having atriglyceride-lowering effect (e.g., fenofibrate, gemfibrozil,bezafibrate, clofibrate, sinfibrate, clinofibrate and the like).

Hypotensive agents include, for example, angiotensin converting enzymeinhibitors (e.g., captopril, enalapril, delapril, benazepril,cilazapril, enalapril, enalaprilat, fosinopril, lisinopril, moexipril,perindopril, quinapril, ramipril, trandolapril and the like) orangiotensin II antagonists (e.g., losartan, candesartan cilexetil,olmesartan medoxomil, eprosartan, valsartan, telmisartan, irbesartan,tasosartan, pomisartan, ripisartan forasartan, and the like).

Antiobesity agents include, for example, central antiobesity agents(e.g., dexfenfluramine, fenfluramine, phentermine, sibutramine,amfepramone, dexamphetamine, mazindol, phenylpropanolamine, clobenzorexand the like), gastrointestinal lipase inhibitors (e.g., orlistat andthe like), .beta.-3 agonists (e.g., CL-316243, SR-58611-A, UL-TG-307,SB-226552, AJ-9677, BMS-196085 and the like), peptide-basedappetite-suppressing agents (e.g., leptin, CNTF and the like),cholecystokinin agonists (e.g., lintitript, FPL-15849 and the like) andthe like.

Diuretics include, for example, xanthine derivatives (e.g., theobrominesodium salicylate, theobromine calcium salicylate and the like),thiazide formulations (e.g., ethiazide, cyclopenthiazide,trichloromethiazide, hydrochlorothiazide, hydroflumethiazide,bentylhydrochlorothiazide, penflutizide, polythiazide, methyclothiazideand the like), anti-aldosterone formulations (e.g., spironolactone,triamterene and the like), decarboxylase inhibitors (e.g., acetazolamideand the like), a chlorbenzenesulfonamide formulations (e.g.,chlorthalidone, mefruside, indapamide and the like), azosemide,isosorbide, ethacrynic acid, piretanide, bumetanide, furosemide and thelike.

Chemotherapeutic agents include, for example, alkylating agents (e.g.,cyclophosphamide, iphosphamide and the like), metabolism antagonists(e.g., methotrexate, 5-fluorouracil and the like), anticancerantibiotics (e.g., mitomycin, adriamycin and the like),vegetable-derived anticancer agents (e.g., vincristine, vindesine, taxoland the like), cisplatin, carboplatin, etoposide and the like. Amongthese substances, 5-fluorouracil derivatives such as furtulon andneofurtulon are preferred.

Immunotherapeutic agents include, for example, microorganisms orbacterial components (e.g., muramyl dipeptide derivative, picibanil andthe like), polysaccharides having immune potentiating activity (e.g.,lentinan, sizofilan, krestin and the like), cytokines obtained by a geneengineering technology (e.g., interferon, interleukin (IL) and thelike), colony stimulating factors (e.g., granulocyte colony stimulatingfactor, erythropoetin and the like) and the like, among thesesubstances, those preferred are IL-1, IL-2, IL-12 and the like.

Immunosuppressive agents include, for example, calcineurininhibitor/immunophilin modulators such as cyclosporine (Sandimmune,Gengraf, Neoral), tacrolimus (Prograf, FK506), ASM 981, sirolimus (RAPA,rapamycin, Rapamune), or its derivative SDZ-RAD, glucocorticoids(prednisone, prednisolone, methylprednisolone, dexamethasone and thelike), purine synthesis inhibitors (mycophenolate mofetil, MMF,CellCept(R), azathioprine, cyclophosphamide), interleukin antagonists(basiliximab, daclizumab, deoxyspergualin), lymphocyte-depleting agentssuch as antithymocyte globulin (Thymoglobulin, Lymphoglobuline),anti-CD3 antibody (OKT3), and the like.

In addition, agents whose cachexia improving effect has been establishedin an animal model or at a clinical stage, such as cyclooxygenaseinhibitors (e.g., indomethacin and the like) [Cancer Research, Vol. 49,page 5935-5939, 1989], progesterone derivatives (e.g., megestrolacetate) [Journal of Clinical Oncology, Vol. 12, page 213-225, 1994],glucosteroid (e.g., dexamethasone and the like), metoclopramide-basedagents, tetrahydrocannabinol-based agents, lipid metabolism improvingagents (e.g., eicosapentanoic acid and the like) [British Journal ofCancer, Vol. 68, page 314-318, 1993], growth hormones, IGF-1, antibodiesagainst TNF-.alpha., LIF, IL-6 and oncostatin M may also be employedconcomitantly with a compound according to the present invention.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references andpublished patents and patent applications cited throughout theapplication are hereby incorporated by reference.

EXEMPLIFICATION OF THE INVENTION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention,as one skilled in the art would recognize from the teachings hereinaboveand the following examples, that other assays, cell types, agents,constructs, or data analysis methods, all without limitation, can beemployed, without departing from the scope of the invention as claimed.

The contents of any patents, patent applications, patent publications,or scientific articles referenced anywhere in this application areherein incorporated in their entirety.

The practice of the present invention will employ, where appropriate andunless otherwise indicated, conventional techniques of cell biology,cell culture, molecular biology, transgenic biology, microbiology,virology, recombinant DNA, and immunology, which are within the skill ofthe art. Such techniques are described in the literature. See, forexample, Molecular Cloning: A Laboratory Manual, 3rd Ed., ed. bySambrook and Russell (Cold Spring Harbor Laboratory Press: 2001); thetreatise, Methods In Enzymology (Academic Press, Inc., N.Y.); UsingAntibodies, Second Edition by Harlow and Lane, Cold Spring Harbor Press,New York, 1999; Current Protocols in Cell Biology, ed. by Bonifacino,Dasso, Lippincott-Schwartz, Harford, and Yamada, John Wiley and Sons,Inc., New York, 1999; and PCR Protocols, ed. by Bartlett et al., HumanaPress, 2003.

Example 1 Identification of CoQ10 as a MIM

In order to evaluate CoQ10 as a potential MIM, CoQ10 in oxidized formwas exogenously added to a panel of cell lines, including both cancercell lines and normal control cell lines, and the changes induced to thecellular microenvironment profile for each cell line in the panel wereassessed. Changes to cell morphology/physiology, and to cellcomposition, including both mRNA and protein levels, were evaluated andcompared for the diseased cells as compared to normal cells. The resultsof these experiments identified CoQ10 and, in particular, the oxidizedform of CoQ10, as a MIM.

In a first set of experiments, changes to cell morphology/physiologywere evaluated by examining the sensitivity and apoptotic response ofcells to CoQ10. A panel of skin cell lines including a control celllines (primary culture of keratinocytes and melanocytes) and severalskin cancers cell lines (SK-MEL-28, a non-metastatic skin melanoma;SK-MEL-2, a metastatic skin melanoma; or SCC, a squamous cell carcinoma;PaCa2, a pancreatic cancer cell line; or HEP-G2, a liver cancer cellline) were treated with various levels of Coenzyme Q10. The results ofthese experiments demonstrated that the cancer cell lines exhibited analtered dose dependent response as compared to the control cell lines,with an induction of apoptosis and cell death in the cancer cells only.Exemplary experiments are described in detail in Example 3 below.

Assays were next employed to assess changes in the composition of thecell following treatment with CoQ10. Changes in gene expression at themRNA level were analyzed using Real-Time PCR array methodology.Exemplary experiments are described in detail in Examples 6 and 9-13below. In complementary experiments, changes in gene expression at theprotein level were analyzed by using antibody microarray methodology,2-dimensional gel electrophoresis followed by protein identificuationusing mass spectrometry characterization, and by western blot analysis.Exemplary experiments are described in detail below in Examples 4, 7 and8, respectively. The results from these assays demonstrated thatsignificant changes in gene expression, both at the mRNA and proteinlevels, were induced in the cell lines examined due to the addition ofthe oxidized form of CoQ10. Genes modulated by CoQ10 treatment werefound to be clustered into several cellular pathways, includingapoptosis, cancer biology and cell growth, glycolysis and metabolism,molecular transport, and cellular signaling.

Experiments were carried out to confirm the entry of CoQ10 into cellsand to determine the level and form of CoQ10 present in the cells. Inparticular, the level of Coenzyme Q10, as well as the form of CoQ10(i.e., oxidized or reduced), present in the mitochondria was determinedby analyzing mitochondrial enriched preparations from cells treated withCoQ10. The level of Coenzyme Q10 present in the mitochondria wasconfirmed to increase in a time and dose dependent manner with theaddition of exogenous Q10. In a surprising and unexpected result, CoQ10was determined to be present in the mitochondria primarily in oxidizedform. In addition, changes in levels of proteins from mitochondriaenriched samples were analyzed by using 2-D gel electrophoresis andprotein identification by mass spectrometry characterization. Theresults from these experiments demonstrated that the levels of theoxidized form of CoQ10 in the mitochondria over the time course examinedcorrelated with a wide variety of cellular changes, as evidenced by themodulation of mRNA and protein levels for specific proteins related tometabolic and apoptotic pathways. Exemplary experiments are described indetail in Example 5 below.

The results described by Applicants herein identified the endogenousmolecule CoQ10 and, in particular, the oxidized form of CoQ10, as a MIM.For example, the results identified CoQ10 as a MIM, since CoQ10 wasobserved to induce changes in gene expression at both the mRNA andprotein level. The results identified CoQ10 as having multidimentionalcharacter, since CoQ10 induced differential changes in cellmorphology/physiology and cell composition (e.g., differential changesin gene expression at both the mRNA and protein level), in a diseasestate (e.g., cancer) as compared to a normal (e.g., non-cancerous)state. Moreover, the results identified CoQ10 as having multidimensionalcharacter in that CoQ10 was capable of entering a cell, and thusexhibited both therapeutic and carrier effects.

Example 2 Methods for Identifying Disease Relevant Processes andBiomarkers for Metabolic Disorders

From the cell based assays in which cell lines were treated with amolecule of interest, the differences in treated vs non-treated cells isevaluated by mRNA arrays, protein antibody arrays, and 2D gelelectrophoresis. The proteins identified from comparative sampleanalysis to be modulated by the MIM or Epi-shifter, are evaluated from aSystems Biology perspective with pathway analysis (Ingenuity IPAsoftware) and a review of the known literature. Proteins identified aspotential therapeutic or biomarker targets are submitted to confirmatoryassays such as Western blot analysis, siRNA knock-down, or recombinantprotein production and characterization methods.

Materials and Methods for Examples 3-8

Coenzyme Q10 stock

A 500 μM Coenzyme Q10 (5% isopropanol in cell growth media) was preparedas follows. A 10 mL 500 μM Coenzyme Q10 stock was made fresh every time.

Molecular Weight: 863.34

(0.0005 mol/L)(0.010 L)(863.34 g/mol)=0.004317 g

To make 10 mL of 500 μM stock, 4.32 mg Coenzyme Q10 was weighted out ina 15 mL falcon tube, and 500 μL isopropanol was added. The solution waswarmed in a 50-60° C. water bath while swirling to dissolve completely.To this solution, 9.5 mL of media (the same media in which the cells aregrown) was added.

Cell Culture

Cells were obtained from the American Type Culture Collection or Gibco.Cells were grown in DMEM/F-12 media supplemented with 5% fetal bovineserum, 0.25 ug/mL Amphotericin, 100 ug/mL Streptomycin, and 100 U mL-1penicillin. Cells were maintained in an atmosphere of 95% air and 5% CO2at 37 degrees C.

Coenzyme Q10 Treatment and Total Protein Isolation

Cells were grown to 85% confluency prior to exposure with Q10.Supplemented media was conditioned with Q10 to 50 and 100 micro molarconcentrations. Flasks were treated with control, 50 μM Q10, and 100 μMQ10 in triplicate. Protein was isolated from the treated and controlflask after 4, 8, 12, and 24 hours. For isolation of proteins, cellswere washed three times with 5 mL of ice cold PBS at a pH of 7.4. Thecells were then scraped in 3 mL of PBS, pelleted by centrifuge, andre-suspended in a lysis buffer at pH 7.4 (80 mM TRIS-HCl, 1% SDS, withprotease and phosphotase inhibitors). Protein concentrations werequantified using the BCA method.

Cell Lines

The cell lines listed below were propagated and a cell bank establishedfor each. Large scale production of cells for various assays wereperformed and the material harvested for analysis. In general, when acell specific media was not required for maintenance of cell lines, themedia used for cell growth was DMEMF-12 with 5% serum. Cells weretypically grown to 75-80% confluence (clear spacing) prior to splittingand use in cell assays and standard practice methods followed. Thefollowing cell lines were established for experiments:

SK-MEL-28 (non-metastatic skin melanoma)SK-MEL-2 (metastatic skin melanoma)HEKa (kerantinocytes, skin control)HEMa (melanocyte, skin control)nFIB (neonatal fibroblasts)HEP-G2 (liver cancer) [SBH cell line]SkBr-3 (breast cancer, Her2 overexpressed)MCF-7 (breast cancer, p53 mutation)PC-3 (prostate cancer) [SBH cell line]SkBr-3 (human breast adenocarcinoma)

NCI-ES-0808

SCC (squamous cell carcinoma)

PaCa-2 NIH-3T3 Cell Culture:

Cells were obtained for the American Type Culture Collection or Gibco.Cells were grown in DMEM/F-12 media supplemented with 5% fetal bovineserum, 0.25 ug/mL Amphotericin, 100 ug/mL Streptomycin, and 100 U mL-1penicillin. Cells were maintained in an atmosphere of 95% air and 5% CO2at 37 degrees C.

Skin malignant melanoma SK-MEL28 cells were grown and maintained inDMEM/F12 with Glutamax (Invitrogen, Carlsbad Calif.) supplemented with5% FBS, amphotericin and penicillin/streptomycin. Cells were grown at37° C. with 5% CO₂. Details of additional cell line and growthconditions are outlined in the table below.

TABLE 1 Cell lines analyzed for sensitivity to Q10. Cell LineDescription Growth Conditions PaCa2 Pancreatic Carcinoma DMEM/F12 withGlutamax + 10% FBS, 2.5% Horse Serum, amphotericin, penicillin/streptomycin. HepG2 Hepatocellular MEM with Earles Salts supple-Carcinoma mented with 10% FBS, amphotericin, penicillin/ streptomycin,sodium pyruvate and non-essential amino acids. PC3 Prostate DMEM/F12with Glutamax, Adenocarcinoma supplemented with 5% FBS, amphotericin andpenicillin/streptomycin. SKBr3 Breast Cancer DMEM/F12 with Glutamaxsupplemented with 5% FBS and amphotericin, penicillin/streptomycin.MCF-7 Breast Cancer DMEM/F12 with Glutamax supplemented with 5% FBS andamphotericin, penicillin/streptomycin.

Q10 Treatment of SKMEL28 Cells:

SK-MEL28 cells were treated with 100 μM Q10 or the control vehicle. Theformulation of the Q10 was as follows. In a 15 mL capped tube, 4.32 mgof Q10 (supplied by Cytotech) was transferred and then dissolved by theaddition of 500 μL of isopropanol. The resulting solution was warmed ina 65° C. water bath and vortexed at high speed. The Q10/isopropanolsolution was made to a volume of 10 mL with the addition of equilibratedcell culture media. The stock solution was then vortexed to ensuremaximum solubility of Q10. The stock solution was diluted (2 mL of stockwith 8 mL of media) to obtain a final concentration of 100 μM Q10. Forthe control vehicle, 9.5 mL of media was added to 500 μL of isopropanol.The control stock was further diluted (2 mL of stock) with 8 mL ofmedia. Cells were harvested 6, 16, 24, 48 or 72 hours after the start ofthe treatment.

Q10 Treatment of SCC Cells:

SCC cells were treated with 100 μM Q10 (prepared as described above)either for 6 hours or 24 hours. The control cells were untreated cells.Cells were harvested and pelleted at the different times after treatmentand the pellets were flash frozen and stored at −80° C. until the RNAwas isolated at XTAL as described below.

RNA Isolation:

Cells were lysed for RNA isolation at different treatment times usingthe RNeasy Mini kit (Qiagen, Inc., Valencia Calif.) kit following themanufacturer's instructions. RNA was quantified by measuring OpticalDensity at 260 nm.

First Strand Synthesis:

First strand cDNA was synthesized from 1 μg of total RNA using the RT2First Strand Synthesis kit (SABiosciences, Frederick Md.) as permanufacturer's recommendations.

Real-Time PCR:

Products from the first strand synthesis were diluted with water, mixedwith the SYBR green master mix (SABiosciences, Frederick Md.) and loadedonto PCR arrays. Real time PCR was run on the PCR Arrays (ApoptosisArrays, Diabetes Arrays, Oxidative stress and Antioxidant defense Arraysand Heat Shock Protein Arrays.) (SABiosciences, Frederick Md.) on aBiorad CFX96.

Determining Cell Line Sensitivity to Coenzyme Q10 by Nexin Assay forApoptosis:

The percentage of cells in early and late apoptosis was quantifiedfollowing 24 hours of Coenzyme Q10 treatment. Early and late apoptosiswas used as a marker to understand the differences in sensitivity ofvarious cancer cell lines to Coenzyme Q10. The different cell linestested were PaCa2, HepG2, PC-3, SKBr3, MCF-7 and SK− MEL28. Cells wereallowed to adhere overnight in 96-well plates. These cells were treatedwith either control vehicle, 50 μM Q10 or 100 μM Coenzyme Q10. After 24hours, the presence of apoptotic cells was estimated on a PCA96 flowcytometer (Guava Technologies, Hayward, Calif.). In addition, some cellswere treated with 4 μM Staurosporine for 2 hours as a positive controlfor apoptosis. Cells were first washed with PBS and detached with 50 μLof Accumax (Innovative Cell Technologies, San Diego, Calif.) at roomtemperature. The dissociation was stopped by addition of culture mediumcontaining 1% Pluronic F-68 (Sigma-Aldrich, St. Louis, Mo.). Then 100 μLof Nexin reagent (Guava Technologies, Hayward, Calif.) was added to eachof the wells. After 20 minutes of incubation in the dark, the assay wasperformed in low binding plates to minimize reattachment of cells to thesubstrate. The Nexin Reagent contains two dyes. Annexin-V-PE whichdetects phosphotidyl serine on the outside of a cell; a characteristicof early apoptotic cells. The second dye, 7-AAD permeates only lateapoptotic cells while being excluded from live (healthy) and earlyapoptotic cells. The percentage of four populations of cells; live,early apoptotic, late apoptotic and debris was determined using theCytosoft 2.5.7 software (Guava Technologies, Hayward, Calif.).

Immunoblotting

Approximately 50 μg of protein were assayed per sample byimmunoblotting. All treatments were run in triplicate with controls.Proteins were separated on 12% TRIS-HCl gels, transferred viaelectrophoresis to nitro-cellulose membranes and blocked using a 5% milkand TBST solution prior to incubation with primary antibodies. Theprimary antibodies were incubated overnight at 4 degrees C. in a 5% BSAand TBST solution. Secondary antibodies were incubated for one hour at 4degrees. All antibodies were purchased from Cell Signaling Technology.Antibodies were used at a ratio of 1:1000, with the exception of PActinat a ratio of 1:5000. Blots were developed and results were quantifiedusing the NIH Java based densitometer analysis software Image J. Allblots were also probed for and normalized to their respective PActinexpression.

Two-Dimensional Electrophoresis

Before isoelectric focusing (IEF), samples were solubilized in 40 mMTris, 7 M urea, 2 M thiourea, and 1% C7 zwitterionic detergent, reducedwith tributylphosphine, and alkylated with 10 mM acrylamide for 90 minat room temperature. After the sample was run through a 10-kDa cutoffAmicon Ultra device with at least 3 volumes of the resuspension buffer,consisting of 7 M urea, 2 M thiourea, and 2% CHAPS to reduce theconductivity of the sample. One hundred micrograms of protein weresubjected to IEF on 11-cm pH 3 to 10, pH 4 to 7 or pH 6 to 11immobilized pH gradient strips (GE, Amersham, USA) to 100,000 voltshour. After IEF, immobilized pH gradient strips were equilibrated in 6 Murea, 2% SDS, 50 mM Tris-acetate buffer, pH 7.0, and 0.01% bromphenolblue and subjected to SDS-polyacrylamide gel electrophoresis on 8 to 16%Tris-HCl Precast Gel, 1 mm (Bio-Rad, USA). The gels were run induplicate. They were either fixed, stained in SYPRO Ruby, 80 mL/gel(Invitrogen, USA) and imaged on Fuji FLA-5100 laser scanner ortransferred onto PVDF membrane.

Additional information was obtained for a control sample to test theutility of protein identification through the use of methods thatutilize dPC (Protein Forest Inc.) selective pI fractionation, followedby trypsin digestion of the dPC plug with mass spec identification andsemi-quantization (Nanomate or LC/LTQ/MS). The dPC analysis performedwith a control sample demonstrated its utility in identifying a largesubset of proteins. The materials produced during the studies werearchived so that they may be utilized as a resource should the futureneed arise

2D Gel Image Analysis:

Analysis of all gel images was performed using Progenesis Discovery andPro (Nonlinear Dynamics Inc., Newcastle upon Tyne, UK). After spotdetection, matching, background subtraction, normalization, andfiltering, data for SYPRO Ruby gel images was exported. Pairwisecomparisons between groups were performed using the Student's t test inProgenesis Discovery to identify spots whose expression wassignificantly altered (p>0.05).

Antibody Array:

An antibody microarray (Panorama XP725 Antibody Array, Sigma) wasutilized to screen over 700 protein antibodies to assess changes at theprotein concentration level in Q10 treated cells (SK-MEL-28, SCC). Theexpression of a protein in a cell extract is detected when it is boundby a corresponding antibody spotted on the slide. Prior to binding, theproteins are directly labeled with a fluorescent dye which is used forfluorescent visualization and quantitative analysis. The array is usedfor comparing protein expression profiles of two samples (test versusreference samples), each labeled with a different CyDye (Cy3 or Cy5) andthe two samples are applied simultaneously at equal proteinconcentrations on the array. Fluorescent signal intensity for eachsample is then recorded individually at the wavelength corresponding tothe dye label of the sample and compared.

High doses of Coenzyme Q10 regulates expression of genes involved in theapoptotic, diabetic and oxidative stress pathways in cultured SKMEL-28cells. Experimental details: SKMEL-28 cells (ATCC Catalog #HTB-72) arenon metastatic, skin melanoma cells that were cultured in DMEM-F12containing Glutamax (Invitrogen Cat# 10565-042) supplemented with 5%FBS, Penicillin, Streptomycin and Amphotericin, were treated with thevehicle or 100 uM Coenzyme Q10 for varying amounts of time. Any changesin gene expression consequent to Coenzyme Q10 treatment were quantifiedusing Real time PCR Arrays (Apoptosis Cat #PAHS-12, Diabetes Cat#PAHS-023 and Oxidative Stress Cat #PAHS-065). (SABiosciences,Frederick, Md.).

A stock concentration of 500 uM Coenzyme Q10 was prepared by dissolving4.32 mg in 500 ul of isopropanol which was further diluted to 10 ml byaddition of media. Alternate vortexing and heating to 65° C. dissolvedthe Coenzyme Q10. 2 ml of the stock solution was diluted to 10 ml withmedia to get a 100 uM Q10 containing media that was used to treat cells.A vehicle was prepared in parallel with a similar protocol except thatthe Coenzyme Q10 was not added.

SKMEL-28 cells were plated at a density of 1×10⁵ cells/well in a 6-wellplate. After 24 hours, when cells had attached and were at 50%confluence, either the vehicle or 100 uM Q10 was added. Cells wereharvested by at 6, 16, 24, 48 or 72 hours after Q10 treatment while thevehicle treated cells were harvested after 24 hours. Cells were lysedfor RNA isolation at different treatment times using the RNeasy Mini kit(Qiagen, Inc., Valencia Calif. Cat #74104) kit following themanufacturer's instructions using a spin column and on-column DNasetreatment. RNA was quantified by measuring absorbance at 260 nm.

Real time PCR was preceded by first strand cDNA synthesis using 0.4-lugof total RNA as the template using the RT2 First Strand Synthesis kit(SABiosciences, Frederick Md. Cat# C-03) with a genomic DNA eliminationstep as per manufacturer's recommendations. Products from the firststrand synthesis were diluted with water, mixed with the SYBR greenmaster mix (SABiosciences, Frederick Md. Cat#PA-010-12) and loaded ontoPCR arrays that contain primer assays for 84 different genes linkedwithin a common pathway, 5 housekeeping genes used for normalization,reverse transcription and PCR controls. Real time PCR was run on aBiorad Cfx96. The amplification was initiated with a hot start toactivate the enzyme, followed by 40 cycles each of (95° C.-15 seconddenaturation step and 60° C.-1 minute annealing and extension step)followed by a melting curve program. Ct values, the output from the PCRthermocycler for all treatment groups were organized on an excelspreadsheet and loaded onto the comparative analysis software availableat http://www.sabiosciences.com/pcdarrayanalysis.php.

Purification of Mitochondria Enriched Samples:

Experimental details: SKMEL-28, NC1-ES0808 and NIH-3T3 cells that weretreated with 100 μM Q10 for 24 or 48 hours along with cells that wereharvested at t=0 were harvested by washing and scraping from T160flasks. Cells were centrifuged, pelleted, flash frozen and stored at−80° C. until the mitochondria were isolated. Cell pellets were thawed,resuspended and ruptured in Dounce homogenizer. The homogenate wascentrifuged and mitochondria were isolated using reagents and theprotocol recommended by the Mitochondria Isolation kit for Culturedcells (MitoSciences, Eugene Oreg., Cat # MS852). The mitochondrialfraction was aliquoted and stored at −80° C.

Coenzyme Q10 and Ubiquinol-10 Quantification Method:

A method for the simultaneous determination of Coenzyme Q10 (Q10) andthe reduced form ubiquinol-10 (Q10H2) was implemented based upon arecently published method (Ruiz-Jimenez, 2007, J. Chromatogr. A, 1175,242-248) through the use of LC-MS/MS with electrospray ionization (ESI)in the positive ion mode. The highly selective identification andsensitive quantitation of both Q10 and Q10H2 is possible, along with theidentification of other selected lipids. An aliquot of the mitochondrialenriched samples from SK-MEL-28 treated with 100 μM Q10 was subjected toa conventional pre-treatment based on protein precipitation (100 μl ofpacked cells sonicated in 300 μl of 1-propanol), liquid-liquidextraction (add 100 μl of water to supernatant and extract X3 with 200μl of n-hexane), evaporation of combined hexane extracts to dryness andreconstitution in 50 μl of 95:5 methanol/hexane (v/v). Analysis was byLC-MS/MS on a Waters Quattro II triple quadrupole mass spectrometer witha Prism RP 1×100 mm, 5 μm particle size column (Keystone Scientific).Isocratic elution with 4 mM ammonium formate in 20% isopropyl alcohol80% methanol at a flow rate of 50 μl/min Ten μl of each sample wasinjected. MRM analysis was performed using m/z 882.7>197.00 (Q10H2) andm/z 880.80>197.00 (Q10) transitions with cone voltage of 40 andcollision energy of 30.

Example 3 Sensitivity of Cell Lines to CoQ10

A number of cell lines were tested for their sensitivity to Q10 after 24hours of application by using a reagent (Nexin reagent) that contains acombination of two dyes, 7AAD and Annexin-V-PE. The 7AAD dye will enterinto cells with permeabilized cell membranes; primarily those cells thatare in late apoptosis. Annexin-V-PE is a dye that binds to Phosphotidylserine, which is exposed on the outer surface of the plasma membrane inearly apoptotic cells. The Nexin reagent thus can be used todifferentiate between different populations of apoptotic cells in a flowcytometer.

PaCa2 cells showed an increase in both early and late apoptotic cells(between 5-10% of gated cells) with 50 μM Q10 and 100 μM Q10 after 24hours of Q10 application. PC-3 cells also showed an increase in bothearly and late apoptotic population with 50 μM and 100 μM Q10, althoughthe increase was less when compared to PaCa2 cells. MCF-7 and SK-MEL28cells showed an increase only in early apoptotic population with 50 μMand 100 μM Q10. HepG2 cells were also sensitive to 50 μM Q10 treatment,where there was an increase of about 20% of the gated populated in thelate apoptotic and early apoptotic stages. SKBr3 was the only cell linetested that did not show any significant increases of early and lateapoptosis with either 50 μM or 100 μM Q10 treatment. The results aredepicted in FIGS. 1-6.

To provide additional confirmation that Q10 treatment causes anapoptotic response in HepG2 liver cancer cells, a second apoptosis assaywas evaluated using the ApoStrand™ ELISA based method that measuressingle-stranded DNA. The ApoStrand™ ELISA is based on the sensitivity ofDNA in apoptotic cells to formamide denaturation and the detection ofthe denatured DNA with a monoclonal antibody to single-stranded DNA(ssDNA). Treatment of the liver cancer cell line HepG2 with 50 and 100μM Q10 resulted in detectable apoptosis, with a dose-response of 17% and32%, respectively (FIG. 7). These results are consistent with theobservation of Q10 inducing apoptosis in other cancer cell lines fromother tissues (e.g., SCC, SKMEL-28, MCF-7, and PC-3).

Example 4 Proteomic Analysis of Cells Treated with Q10

Cell pellets of samples treated with Q10 were analyzed using proteomicmethods. The cell pellets were lysed and treated for use in 2-D gel andWestern blot analysis. Three cell types (SKMEL-28, SCC, and nFib) weretreated with Q10 and submitted to proteomic characterization by 2-D gelelectrophoresis.

Proteomic Analysis of SKMEL-28 Cells Treated with Q10

The first experimental set processed and evaluated by Western blot and2-D gel electrophoresis was the skin cancer cell line SKMEL-28. Thisexperimental set involved SK-MEL-28 cells treated at 3, 6, 12, and 24hours with 0, 50 or 100 μM Q10.

The set of Q10 treated SK-MEL-28 samples were subjected to 2-D gelelectrophoreses (FIG. 8) and were analyzed to identify protein-levelchanges relative to the control samples. A comparative analysis of 943spots across all twenty-four gels was performed, comparing the controlsample against all of the treated samples. The analysis included theidentification of spot changes over the time course due to increase,decrease, or post-translational modification.

The analysis found thirty-two statistically significant differentialspot changes. From this, twenty non-redundant spots were excised andsubmitted for protein identification by trypsin digestion and massspectrometry characterization. The characterized peptides were searchedagainst protein databases with Mascot and MSRAT software analysis toidentify the protein (Table 2).

TABLE 2 Proteins identified to have a differential response to Q10treatment in SKMEL-28 cell. Q10 Time Conc. 2D (hr) (uM) Spot #Expression Difference Protein Name Type 3 50 528 down 1.234 cathepsin DCTSD peptidase 3 50 702 down 1.575 chaperonin containing CCT3 otherTCP1, subunit 3 3 50 74 down 1.383 eukaryotic translation EIF3Gtranslation initiation factor 3 regulator 3 50 829 down 1.074 Ribosomalprotein P2 RPLP2 other 3 50 368 down 1.121 transaldolase 1 TALDO1 enzyme6 50 452 up −1.464 eukaryotic translation EIF6 translation initiationfactor 6 regulator 6 50 175 up −1.32 Stomatin; HSPC322 STOM other 6 50827 up −1.457 Tyrosine 3/Tryptophan YWHAZ enzyme 5-monooxygenaseactivation protein 6 50 139 up −1.628 Vimentin VIM other 6 50 218 up−1.416 Vimentin VIM other 6 50 218 up −1.212 Vimentin VIM other 6 50 139up −1.036 Vimentin VIM other 6 50 507 down 1.379 Lamin B1 LMNB1 other 650 571 down 1.832 mitochandrial import TOMM22 transporter receptor Tom2212 50 166 up −1.171 ALG-2 interacting PDCD6IP other protein 1 12 50 550up −1.747 peptidylprolyl PPIA enzyme isomerase A 12 50 613 down 1.802galectin-1 LGALS1 other 12 50 242 down 1.373 Phosphoglycerate mutase;PGAM2 phosphatase Posphomannomutase 2 24 50 326 down 1.385 glycyl-tRNAsynthase GARS enzyme 24 50 419 down 1.451 Mago-nashi homolog MAGOH other3 100 528 down −1.036 cathepsin D CTSD peptidase 3 100 702 down 1.151chaperonin containing CCT3 other TCP1, subunit 3 3 100 74 down 1.122eukaryotic translation EIF3G translation initiation factor 3 regulator 3100 829 down 1.145 Ribosomal protein P2 RPLP2 other 3 100 368 down 1.209transaldolase 1 TALDO1 enzyme 6 100 139 up −1.829 Vimentin VIM other 6100 218 up −1.761 Vimentin VIM other 6 100 452 down 1.134 eukaryotictranslation EIF6 translation initiation factor 6 regulator 6 100 252down 1.4 Sec 13 protein, ? Keratin II 6 100 827 down 1.12 Tyrosine3/Tryptophan YWHAZ enzyme 5-monooxygenase activation protein 12 100 76up −1.679 galectin-1; keratin II LGALS1 other

A key finding in this experiment was the decrease of Transaldolase 1,which supports the premise that Q10 acts by altering the metabolic statewithin the cancer cell. Transaldolase 1 is an enzyme in the pentosephosphate pathway (also known as the hexose monophosphate shunt).Transaldolase (EC:2.2.1.2) catalyses the reversible transfer of athree-carbon ketol unit from sedoheptulose 7-phosphate to glyceraldehyde3-phosphate to form erythrose 4-phosphate and fructose 6-phosphate. Thisenzyme, together with transketolase, provides a link between theglycolytic and pentose-phosphate pathways. This is relevant tonucleotide and NADPH synthesis, to facilitate production of reducingequivalents for biosynthetic reactions and maintenance of a reducingenvironment.

A recent publication (Basta, P., et. al. August 2008, Cancer DetectPrevention, 32, 200-208) provided evidence of genetic polymorphism inTransaldolase and was linked to squamous cell carcinoma of the head andneck. Another recent publication (Qian, Y., et. al. May 2008, Biochem J,415, 123-134) identified transaldolase deficiency as a modulator ofmitochondrial homoeostasis, Ca2+ fluxing and apoptosis.

From these initial results, the other proteins identified by 2-D gelelectrophoresis as being modulated by Q10 in SK-MEL-28 were analyzed forknown relationships (FIG. 9). A functional evaluation of these proteinsrevealed that there was a group involved in 14-3-3-mediated signaling(PDCP61P, YWHAZ, and VIM), along with individual proteins linked to avariety of processes [cell cycle; pentose phosphate pathway (TALDO1);ceramide signaling (CTSD); aminoacyl-tRNA biosynthesis (GARS), andmitochondrial protein import (TOM22)].

Proteomic Analysis of SCC Cells Treated with Q10

Another skin cancer cell line, Squamous Cell Carcinoma (SCC), was alsoprepared and analyzed by 2-D gel electrophoreses as a follow-upexperiment the previous SK-MEL-28 analysis The SCC cells were treatedwith 100 μM Q10 for 6 hour or 24 hours before harvesting. A control ofuntreated cells was also harvested. The cell pellets were lysed and thesamples were subjected to 2-D electrophoresis (in duplicate). Analysisof over six hundred protein spots in the comparative study wasperformed, comparing the control sample against the six hour andtwenty-four hour treatments.

The top twenty-five statistically significant differential spot changeswere evaluated from the comparative analysis of the 2-D electrophoresisgels. From this, twelve spots were excised and submitted foridentification by trypsin digestion and mass spectrometrycharacterization (results summarized in Table 3 below).

TABLE 3 Proteins identified to have a differential response to 100 μMQ10 treatment in SCC cells at 6 and 24 hours. Spot Cellular Response #Protein Name localization Function (fold change) 331 Transaldolase 1TALDO1 Cytoplasm Enzyme Decrease (1.5) at 6 and 14 hr 23 Human BSCvC20ORF3 Plasma strictosidine Decrease (chromosome 20 membrane synthase(2.1) at 6 and reading frame 3) 24 hr 54 NM23 protein NME1 Nucleus,Kinase Increase (mitochondria?) (−1.2) at 6 hr, decrease at 24 hr 116two Human ESTs HSP70 Decrease from MCF7 (2.6) at 6 hr, breast cancercell further decrease line (HSP 70) at 24 hr 176 Heat shock HSPB1Cytoplasm Response to Increase 27 kDa protein 1 environmental (−1.9) at6 and stresses 24 hr 135 Keratin I KRT1 Cytoplasm intermediate Decreasefilaments (2.3) at 6 and 24 hr 50 Keratin 14 KRT14 Cytoplasmintermediate Increase filaments (−1.6) at 6 and 24 hr 68 Keratin 13KRT13 Cytoplasm intermediate Increase filaments (−1.5) at 6 and 24 hr 49Proteasome PSMB7 Cytoplasm Proteasome Decrease Beta 7 subunit (1.6) at24 hr only 93 Proteasome PSME3 Cytoplasm peptidase Decrease activator(1.3) at 24 hr subunit 3 only 66 Rho GDP ARHGDIA Cytoplasm InhibitorDecrease dissociation (1.5) at 6 hr inhibitor (GDI) only alpha 1Unknown? Decrease (9.5)

Transaldolase 1: As previously observed in the SKMEL-28 cells treatedwith Q10, the enzyme Transaldolase 1 was modulated with a decrease inlevels. This provides an independent confirmation of the previouslyobservation of a linkage between Q10 and alterations in transaldolase(and thus the metabolic state of the cell).

Transaldolase is an enzyme in the non-oxidative phase of the pentosephosphate pathway (FIG. 10). The pentose phosphate pathway is criticalin the metabolic state of cells for the generation of nicotinamideadenine dinucleotide phosphate (reduced NADH), for reductivebiosynthesis, and in the formation of ribose which is an essentialcomponent of ATP, DNA, and RNA. Transaldolase also links the pentosephosphate pathway to glycolysis. Glycolysis is the metabolic pathway bywhich cancer cells obtain the energy needed for cell survival, as themitochondrial process of oxidative phosphorylation is not utilized. Q10is an essential coenzyme factor required for oxidatative phosphorylationand mitochondrial ATP production.

BSCv: Spot 23 was a novel human protein from Chromosome 20 named BSCv.BSCv protein is also known as Adipocyte plasma membrane-associatedprotein (Gene names: APMAP or C20orf3) and is predicted to be asingle-pass type II membrane protein with sequence similarity to thestrictosidine synthase family of proteins. Q10 treatment caused areduction in the levels of this protein. This protein is not wellcharacterized, nor has its homology with strictosidine synthases beenconfirmed. Interestingly, this protein has been associated with a rolein adipocyte differentiation (Albrektsen et al., 2001). Recent proteomicstudies of human omental adipose tissue identified BSCv as one of nineproteins with differential expression for polcystic ovary syndrome(PCOS) from morbidly obese women (Corton, 2008 Hum. Reprod. 23:651-661). As a cell surface protein that responds to Q10, an antibodyagainst BSCv would be useful as a biomarker. Based on the currentresults and the literature available, BSCv may a have a potential rolein cancer and diabetes.

NM23A: Non-metastatic cells 1, protein (NM23A, also known as NME1) isthought to be a metastasis suppressor. This gene (NME1) was identifiedbecause of its reduced mRNA transcript levels in highly metastaticcells. The protein has activity as a nucleoside diphosphate kinase (NDK)and exists as a hexamer composed of ‘A’ (encoded by this gene) and ‘B’(encoded by NME2) isoforms. Mutations in this gene have been identifiedin aggressive neuroblastomas. NDK activities maintain an equilibriumbetween the concentrations of different nucleoside triphosphates suchas, for example, when GTP produced in the citric acid (Krebs) cycle isconverted to ATP. The NDK complex is associated with p53 throughinteraction with STRAP. It is noteworthy that STRAP is linked to HNF4A.Thus, NM23A is a potential protein involved in pathways important forcell control and disease treatment.

Rho GDP dissociation inhibitor (GDI) alpha: GDI Regulates the GDP/GTPexchange reaction of the Rho proteins by inhibiting the dissociation ofGDP from them, and the subsequent binding of GTP to them. The protein isupregulated in cancer cells.

Example 5 Mitochondrial Enrichment Analysis

Several lines of evidence suggested that a closer evaluation of the roleof mitochondrial proteins and cancer biology and Q10 response waswarranted. First, there is the essential role of Q10 in themitochondrial oxidative phosphorylation process for energy production innormal cells. However, the metabolic shift that occurs in cancer cellsis to energy production through the alternative pathway of glycolysis,which does not require Q10. Second, the apoptotic response of cellsrequires mitochondrial proteins to occur. Q10 has been established asstimulating apoptosis in cancer cells (Bcl-2 family proteins, cytochromec). Finally, new mitochondrial proteins were identified as beingmodulated by Q10 treatment, as exemplified by the modulation in proteinlevels of the mitochondrial import receptor protein TOM22 (seeexperiments described herein).

Production of Mitochondrial Enriched Samples

The skin cancer SKMEL-28 cells were treated with 100 μM Q10 or a mockvehicle for 6, 19, or 48 hours. The cells were harvested by washing andscraping the cells from T-160 flasks (4 for each time point). The cellswere collected by centrifugation and the pellets flash frozen and storedat −80° C. The cell pellets were resuspended and ruptured using a 2 mLDounce homogenizer. The reagents and method were obtained from aMitochondria Isolation Kit for Cultured Cells (MitoSciences, Cat#MS852). The resultant mitochondria samples were divided into 75 μLaliquots (4-5 aliquots per sample) and stored at −80° C.

Proteomic Analysis of Mitochondria Enriched Samples Isolated fromSK-MEL-28 Cells Treated with Q10

2-D gel electrophoresis was performed on proteins solubilized from twoaliquots of the SK-MEL-28 mitochondria enriched samples treated with 100μM Q10 for 6, 19, and 48 hours (along with the corresponding mockvehicle controls). The samples were subjected to 2-D electrophoresis (induplicate). Analysis of 525 protein spots in the comparative study wasperformed, comparing the control samples against the other time pointsamples (FIG. 11).

The nine statistically significant differential spot changes wereselected from the comparative analysis of the 2-D electrophoresis gels.From these, 9 spots were excised and submitted for identification bytrypsin digestion and mass spectrometry characterization

TABLE 4 Proteins identified to have a differential response to Q10treatment in SKMEL-28 mitochondria. Spot Response (fold # Protein NameFunction change) 11 Unknown protein ? ? Up (1.3) at 6 hr, drop to lowlevels after this 131 Unknown, same as ? ? Down (1.3) at 6 hr, spot #11,modified drops more for 19 and 48 hr 279 acyl-CoA thioesterase ACOT7Cleaves fatty acyl- Down (1.3) at 6 hr, 7 isoform hBACHb CoA's into freefatty back to normal at 48 acids and CoA hr 372 Pyruvate kinase PKM2catalyzes the Up (1.5) at 6 hr, back production of to normal at 48 hrphosphoenolpyruvate from pyruvate and ATP 110 ER60 protein PDIA3 Proteindisulfide Up at 19 and 48 hr isomerase 185 Keratin 10 KRT10 intermediatefilament Up only at 19 hr 202 Beta-Actin Structural protein Up only at19 hr 246 Malectin MLEC carbohydrate-binding Up only at 19 hr protein ofthe endoplasmic reticulum and a candidate player in the early steps ofprotein N-glycosylation 75 Coiled-coil domain CCDC58 Conservedhypothetical Up at 48 hr containing 58 protein- nuclear pore forming

Acyl-CoA thioesterase 7: Acyl-CoA thioesterase 7 (ACOT7) is a member ofthe enzyme family that catalyzes the hydrolysis of fatty acyl-CoA tofree fatty acid and CoA. This enzyme thus has a role in the regulationof lipid metabolism and cellular signaling. ACOT7 has a preference forlong-chain acyl-CoA substrates with fatty acid chains of 8-16 carbonatoms (C8-C16). The exact cellular function is ACOT7 is not fullyunderstood. The transcription of this gene is activated by sterolregulatory element-binding protein 2, thus suggesting a function incholesterol metabolism.

The results in this Example indicate that ACOT7 is potentially involvedin the metabolism of Q10, either directly or indirectly. Thus, targetingACOT7 could facilitate modulation of intercellular levels of Q10 andthus impact cellular Q10 effects.

Pyruvate kinase: Pyruvate kinase is an enzyme involved in the last stepof glycolysis. It catalyzes the transfer of a phosphate group fromphosphoenolpyruvate (PEP) to ADP, yielding one molecule of pyruvate andone molecule of ATP.

The protein is presumably that of PKM2, the type 2 isoform, as this wasidentified from the mitochondria enriched SK-MEL-28 sample. This isoformis well known to be involved in tumor cell formation and regulation.

Quantification of Q10 Levels in Mitochondria

A method for the simultaneous determination of Coenzyme Q10, (Q10) andthe reduced form ubiquinol-10 (Q10H2) was implemented based upon arecently published method (Ruiz-Jimenez, 2007, J. Chroma A, 1175,242-248) through the use of LC-MS-MS with electrospray ionization (ESI)in the positive mode. The highly selective identification and sensitivequantitation of both Q10 and Q10H2 is possible, along with theidentification of other selected lipids. An aliquot of the mitochondrialenriched samples from SK-MEL-28 treated with 100 μM Q10 were subject toa conventional pre-treatment based on protein precipitation,liquid-liquid extraction, evaporation to dryness and reconstitution with95:5 methanol/hexane (v/v).

In this analysis, Q10, Q10H2, and Q9 were quantitated (Table 5). Thelevels of the related molecule Q9 were low, and near the level ofdetection. The level of the untreated samples were relativelyconsistent, with the 6 hour Q10 treated sample having this same level.To control for sample variance in total material, the levels ofcholesterol was also measured to confirm that the differences were notdue to sample size errors. When the Q10 levels were corrected againsttotal protein values obtained by protein extraction other aliquots ofthe same mitochondrial preps, the relative ratios were comparative.Thus, a significant increase in Q10 levels was obtained at 19 hours(˜3-fold) with an even larger increase by the 48 hour time point(˜6-fold) (FIG. 12).

TABLE 5 HPLC-MS Quantification results for the levels of Q10 present inmitochondrial enriched samples from SK-MEL-28 cells treated with 100 μMQ10 in the media. Peak Area ng/Sample μg/sample File Sample Injection Q9Q10 Q9 Q10 Q10H₂ Cholesterol 081204-05 100 ng Std 245,342 352792081204-06 6 hr mock#1 10% 2560 32649 1.04 9.25 081204-07 Solvent Blank#15 ul 3781 3174 1.54 0.9 081204-08 Solvent Blank#2 5 ul 2396 4399 0.981.25 081204-09 6 hr mock#2 20% 1572 36328 0.64 10.3 081204-10 SolventBlank#3 10 ul  1722 2504 0.7 0.71 081204-11 48 hr Q10 treated 20% 4879164496 1.99 46.63 0.28 13.86 081204-12 48 hr mock 20% 2412 25552 0.987.24 0.09 13.04 081204-13 6 hr Q10 treated 20% 692 25427 0.28 7.21081204-14 19 hr Q10 treated 20% 1161 59164 0.47 16.77 081204-15 19 hrmock 20% 901 19999 0.37 5.67

A surprising result from this study was the finding that the Q10 wassupplied to the cells as the oxidized form. For the 48 hour samples, thereduced form Q10H2 was also measured and found to be present insignificantly lower amounts (0.28 ng/sample of CoQ10H2 as compared to46.63 ng/sample of CoQ10). There was a general increase (3-fold) in thelevels of Q10H2 in the Q10 treated 48 hour sample, although the levelswere near the presumed detection limit of the assay. Interestingly, theoxidized form (Q10) can act as a pro-oxidant in biological systems.According to the literature, when human plasma was evaluated for Q10 andQ10H2, the majority (90%) of the molecule was found in the reduced formof Q10H2 (Ruiz-Jimenez, 2007, J. Chroma A, 1175, 242-248) which can actas an anti-oxidant.

Thus, these results confirm and quantitate that the levels of Q10increase in the mitochondria upon the exogenous addition of Q10 to themedia. A surprising and unexpected discovery was that Q10 was maintainedin the supplied oxidized form (pro-oxidant) and not converted to thereduced (anti-oxidant) form of Q10H2 in any significant amounts.

Example 6 Real-Time PCR Arrays Experiment 1 Apoptosis Array

As discussed above in Example 3, exposure of cancer cells to Q10 inducesa portion of these cells to die due to apoptotic processes. To identifyproteins that were involved in the Q10 response, real-time polymerasechain reaction (RT-PCR) methods were employed to identify changes in thelevel of mRNA for genes/proteins involved in targeted pathway arrays forapoptosis.

Using PCR arrays as a screening tool, a spectrum of molecular targetsthat would potentially offer an insight to the mode of biological actionof Q10 within the cells were thus evaluated. Changes in mRNA levels wereevaluated using real-time PCR quantification to assess mRNA levels inpre-selected subsets containing 80 pathway specific targets.

For the interpretation of mRNA results, the genes that were altered intheir mRNA transcription by a two-fold level were identified andevaluated. The level of gene transcription to produce mRNA only providesa rough estimate of potential changes in the level of the expressedprotein. The skilled artisan will appreciate that each mRNA may havedifferent rates at which it is degraded or its translationinefficiently, thus resulting in differing amounts of protein.

SkBr-3 Cells Treated with 50 um Q10 for 24 Hours

The assay method of RT-PCR was utilized to provide a measure of mRNAlevel changes to a total of 84 apoptotic pathway related proteins. Theexperiments with the real-time PCR apoptosis analysis on SkBr3 with Q10(24 hr) identified the following mRNA's being affected: Bcl2, Bcl2L1,Bcl2L11, Birc6, Bax, Xiap, Hprt1, Apaf1, Ab11, Braf. These results againprovided supporting evidence for the apoptotic response of cancer cellsto Q10 treatment.

TABLE 6A Up-Down Symbol Regulation Unigene Refseq Description GnameBCL2L1 13.1957 Hs.516966 NM_138578 BCL2-like 1 BCL-XL/S BNIP2 6.3291Hs.646490 NM_004330 BCL2/adenovirus E1B 19 kDa BNIP-2/NIP2 interactingprotein 2 BCL2 5.4717 Hs.150749 NM_000633 B-cell CLL/lymphoma 2 Bcl-2BIRC6 4.7966 Hs.150107 NM_016252 Baculoviral IAP repeat- APOLLON/containing 6 (apollon) BRUCE BCL2L11 4.6012 Hs.469658 NM_006538BCL2-like 11 (apoptosis BAM/BIM facilitator) XIAP 4.3832 Hs.356076NM_001167 X-linked inhibitor of API3/BIRC4 apoptosis BRAF 4.3832Hs.550061 NM_004333 V-raf murine sarcoma viral B-raf 1/ oncogene homologB1 BRAF1 BAX 3.896 Hs.631546 NM_004324 BCL2-associated X protein Baxzeta APAF1 2.6244 Hs.708112 NM_001160 Apoptotic peptidase CED4/DKFZpactivating factor 1 781B1145 HPRT1 −160.6748 Hs.412707 NM_000194Hypoxanthine HGPRT/HPRT phosphoribosyltransferase 1 (Lesch-Nyhansyndrome)

Results that are consistent from three independent experiments fromSK-MEL-28 cells are summarized below in Table 6B. Many genes areregulated in SCC cells as well with 100 μM Q10 treatment. The genes inthe Apoptosis array that appear to be regulated in SCC cells aredescribed in Table 7. We find that many genes are regulated at 6 hours,both in SK-MEL-28 cells and in SCC cells. By 24 hours, the regulation isdecreased. Genes that appear to be regulated in both SK-MEL-28 cells andin SCC cells are described in Table 8.

TABLE 6B Genes in SK-MEL-28 cells regulated by 100 μM Q10 treatment whenanalyzed by the Apoptosis Array. Possible Symbol Description RegulationLocation Functions ABL1 C-abl oncogene 1, Down Regulated NucleusTyrosine Kinase receptor tyrosine at 72 hours kinase BAG1BCL2-associated Up Regulated at Cytoplasm Anti-apoptotic, athanogene 48hours glucocorticoid receptor pathway BCL2 B-cell Down RegulatedCytoplasm Cell death CLL/lymphoma 2 at 48 hours BCL2A1 BCL2-related DownRegulated Cytoplasm Regulates protein A1 at 48 hours Caspases,phosphorylates TP73 BCL2L1 BCL2-like 1 Down Regulated Cytoplasm CaspaseInhibitor at 72 hours BCL2L10 BCL2-like 10 Down Regulated CytoplasmCaspase Activator (apoptosis at 48 hours facilitator) BCL2L11 BCL2-like11 Down Regulated Cytoplasm Pro-Apoptotic, (apoptosis at 48 hoursCaspase3 facilitator) Activator BIRC3 Baculoviral IAP Down RegulatedCytoplasm Anti-apoptotic repeat-containing 3 at 6 hours BIRC8Baculoviral IAP Down Regulated Cytoplasm Activates Caspaserepeat-containing 8 at 48 hours CARD8 Caspase recruitment Down RegulatedNucleus Caspase Activator domain family, at 48 hours member 8 CASP14Caspase 14, Down Regulated Cytoplasm Apoptosis related apoptosis-relatedat 48 hours cysteine peptidase cysteine peptidase CASP5 Caspase 5, DownRegulated Cytoplasm Apoptosis related apoptosis-related at 48 hourscysteine peptidase cysteine peptidase CD40LG CD40 ligand (TNF DownRegulated Extracellular CD40 receptor superfamily, at 48 hours Spacebinding member 5, hyper- IgM syndrome) CIDEA Cell death-inducing UpRegulated at Cytoplasm Pro-Apoptotic DFFA-like effector 48 hours a FADDFas (TNFRSF6)- Down Regulated Cytoplasm Pro-Apoptotic associated viadeath at 6 hours domain FAS Fas (TNF receptor Up Regulated at PlasmaPro-Apoptotic superfamily, 48 hours Membrane member 6) FASLG Fas ligand(TNF Down Regulated Extracellular Pro-Apoptotic superfamily, at 48 hoursSpace member 6) GADD45A Growth arrest and Up Regulated at Nucleus GrowthArrest DNA-damage- 48 hours inducible, alpha HRK Harakiri, BCL2 DownRegulated Cytoplasm Pro-Apoptotic interacting protein at 48 hours(contains only BH3 domain) PYCARD PYD and CARD Down Regulated CytoplasmApoptotic domain containing at 6 hours Protease Activator TNF Tumornecrosis Up Regulated at Extracellular TNF receptor factor (TNF 48 hoursthen Space binding superfamily, down regulated member 2) TNFRSF10A Tumornecrosis Up Regulated at Plasma Caspase Activator factor receptor 48hours then Membrane superfamily, down regulated member 10a TNFRSF10BTumor necrosis Down Regulated Plasma p53 signaling, factor receptor at72 hours Membrane caspase superfamily, activation. member 10b TNFRSF1ATumor necrosis Down Regulated Plasma Pro-apoptotic factor receptor at 72hours Membrane superfamily, member 1A TNFRSF21 Tumor necrosis DownRegulated Plasma Activates Caspase factor receptor at 48 hours Membranesuperfamily, member 21 CD27 CD27 molecule Down Regulated Plasma CaspaseInhibitor at 48 hours Membrane TNFRSF9 Tumor necrosis Down RegulatedPlasma Pro-apoptotic factor receptor at 48 hours Membrane superfamily,member 9 TNFSF10 Tumor necrosis Upregulated at 48 ExtracellularPro-apoptotic factor (ligand) hours Space superfamily, member 10 TP73Tumor protein p73 Down Regulated Nucleus Transcription at 48 hoursfactor TRAF3 TNF receptor- Down Regulated Cytoplasm Zinc-fingerassociated factor 3 at 48 hours domain TRAF4 TNF receptor- DownRegulated Cytoplasm Zinc-finger associated factor 4 at 48 hours domain

TABLE 7 Genes in SCC cells that are regulated by 100 μM Q10 treatmentwhen analyzed by the Apoptosis Array. Symbol Description Regulation.AKT1 V-akt murine thymoma viral oncogene Down regulated at 6 hours andhomolog 1 then up regulated at 24 hours. BAG4 BCL2-associated athanogene4 Up regulated at 24 hours. BAX BCL2-associated X protein Up regulatedat 24 hours. BCL2 B-cell CLL/lymphoma 2 Up regulated at 24 hours. BCL2L1BCL2-like 1 Down regulated at 6 hours and then up regulated at 24 hours.BIRC3 Baculoviral IAP repeat-containing 3 Down regulated at 6 hours.BNIP3 BCL2/adenovirus E1B 19kDa interacting Down regulated at 24 hours.protein 3 CARD6 Caspase recruitment domain family, Down regulated at 6hours. member 6 CASP6 Caspase 6, apoptosis-related cysteine Up regulatedat 24 hours. peptidase CASP7 Caspase 7, apoptosis-related cysteine Upregulated at 24 hours. peptidase CD40 CD40 molecule, TNF receptorsuperfamily Down regulated at 6 hours. member 5 FADD Fas(TNFRSF6)-associated via death Up regulated at 24 hours. domain GADD45AGrowth arrest and DNA-damage-inducible, Up regulated at 24 hours. alphaHRK Harakiri, BCL2 interacting protein (contains Up regulated at 24hours. only BH3 domain) TNFRSF21 Tumor necrosis factor receptor Downregulated at 6 hours. superfamily, member 21 TNFRSF25 Tumor necrosisfactor receptor Down regulated at 6 hours and superfamily, member 25then up regulated at 24 hours. CD27 CD27 molecule Down regulated at 6hours. TNFRSF9 Tumor necrosis factor receptor Down regulated at 6 hours.superfamily, member 9 TNFSF10 Tumor necrosis factor (ligand)superfamily, Up regulated at 24 hours. member 10 CD70 CD70 molecule Downregulated at 6 hours. TP53 Tumor protein p53 Up regulated at 24 hours.TP73 Tumor protein p73 Down regulated at 6 hours and then up regulatedat 24 hours. TRAF2 TNF receptor-associated factor 2 Up regulated at 24hours.

TABLE 8 Genes from the apoptosis array regulated with 100 μM Q10treatment in both SK-MEL-28 and SCC cells. Symbol Description BCL2B-cell CLL/lymphoma 2 BCL2L1 BCL2-like 1 (Bcl-xl) BIRC3 Baculoviral IAPrepeat-containing 3 FADD Fas (TNFRSF6)-associated via death domainGADD45A Growth arrest and DNA-damage-inducible, alpha TNFRSF21 Tumornecrosis factor receptor superfamily, member 21 CD27 CD27 moleculeTNFRSF9 Tumor necrosis factor receptor superfamily, member 9 TNFSF10Tumor necrosis factor (ligand) superfamily, member 10 TP73 Tumor proteinp73 TRAF2 TNF receptor-associated factor 2

Interestingly, the altered mRNA levels showed a significantup-regulation in a series of apoptitic proteins, with Bcl-xl one of thehighest. This was also observed in the protein array experiments onSK-MEL-28 cells.

Bcl-x1 is a transmembrane molecule in the mitochondria (Bcl-x1 standsfor “Basal cell lymphoma-extra large”). It is involved in the signaltransduction pathway of the FAS-L and is one of several anti-apoptoticproteins which are members of the Bcl-2 family of proteins. It has beenimplicated in the survival of cancer cells. However, it is known thatalternative splicing of human Bcl-x mRNA may result in at least twodistinct Bcl-x mRNA species, Bcl-xL and Bcl-xS. The predominant proteinproduct (233 amino acids) is the larger Bcl-x mRNA, Bcl-xL, whichinhibits cell death upon growth factor withdrawal (Boise et al., 1993.Cell 74, 597-608). Bcl-xS, on the other hand, inhibits the ability ofBcl-2 to inhibit cell death and renders cells more susceptible toapoptotic cell death. The employed assays utilized do not distinguishwhich isoform of Bcl-x is being upregulated. The Bcl-x isoform beingupregulated by CoQ10 in these studies may be determined by routinemethods known in the art, e.g., by using RT-PCR methods to evaluate theratio of the two mRNA splicing isoforms (Bcl-xL vs Bcl-sL).

From the survey of apoptotic related proteins it was observed multiplepro- and anti-apoptotic factors were in the BCL-2 family or thatinteract with these factors have modulated expression levels (BCL2L11,BNIP2, BAG1, HRK, BAK1, BCL2, BCL2L1). These proteins governmitochondrial outer membrane permeabilization.

An early marker for apoptotic response is observed with the upregulationof Caspase-9 (16 hour) which is consistent with previous observations ofapoptosis with caspase 3/7 proteins. Induction of stress signalingpathways causes release of cytochrome c from mitochondria and activationof apaf-1 (apoptosome), which in turn cleaves the pro-enzyme ofcaspase-9 into the active form. Once intiated caspase-9 goes on tocleave procaspase-3 & procaspase-7 to trigger additional apoptoticpathways.

There is also a consistent linkage to the tumor necrosis factor receptorfamily of proteins being modulated.

A strong down regulation of tumor protein p73 is also noted. Analyses ofmany tumors typically found in humans including breast and ovariancancer show a high expression of p73 when compared to normal tissues incorresponding areas. Recent finding are suggesting that deregulated overexpression of transcription factors within the body involved in cellcycle regulation and synthesis of DNA in mammalian cells (i.e.: E2F-1),induces the expression of p73. The suggestion is that p73 may be anoncoprotein, but may involve different mechanism that the related p53protein. A schematic showing mapping of the apoptosis pathway isprovided in FIG. 13.

SKMEL-28 Cells

From the survey of apoptotic related proteins it was observed multiplepro- and anti-apoptotic factors were in the BCL-2 family or thatinteract with these factors have modulated expression levels (BCL2L11,BNIP2, BAG1, HRK, BAK1, BCL2, BCL2L1). These proteins governmitochondrial outer membrane permeabilization.

An early marker for apoptotic response is observed with the upregulationof Caspase-9 (16 hour) which is consistent with previous observations ofapoptosis with caspase 3/7 proteins. Induction of stress signalingpathways causes release of cytochrome c from mitochondria and activationof apaf-1 (apoptosome), which in turn cleaves the pro-enzyme ofcaspase-9 into the active form. Once intiated caspase-9 goes on tocleave procaspase-3 & procaspase-7 to trigger additional apoptoticpathways.

TABLE 9 Changes in mRNA levels for SKMEL-28 cells treated with 100 μMA10, evaluated by RT-PCR arrays focused around apoptotic pathways. 6 hr16 hr 24 hr 72 hr Refseq Description Symbol Q10 Q10 Q10 Q10 NM_006538BCL2-like 11 BCL2L11 2.13 2.41 1.92 2.51 (apoptosis facilitator)NM_000875 Insulin-like growth IGF1R 1.77 1.09 1.33 1.25 factor 1receptor NM_004048 Beta-2-microglobulin B2M 1.74 1.76 1.58 3.11NM_003921 B-cell CLL/lymphoma 10 BCL10 1.55 1.87 1.48 −3.11 NM_004330BCL2/adenovirus E1B 19 kDa BNIP2 1.46 1.51 1.57 −1.61 interactingprotein 2 NM_005157 C-abl oncogene 1, receptor ABL1 1.42 2.77 −1.22−2.03 tyrosine kinase NM_004323 BCL2-associated athanogene BAG1 1.411.44 −1.61 −2.45 NM_001229 Caspase 9, apoptosis-related CASP9 1.32 3.961.83 1.14 cysteine peptidase NM_003806 Harakiri, BCL2 interacting HRK1.18 4.52 2.73 −1.14 protein (contains only BH3 domain) NM_001924 Growtharrest and DNA-damage- GADD45A 1.07 3.34 1.13 −2.36 inducible, alphaNM_001188 BCL2-antagonist/killer 1 BAK1 1.06 2.73 −1.00 −4.54 NM_004295TNF receptor-associated TRAF4 −1.91 2.63 −1.58 −740.66 factor 4NM_003842 Tumor necrosis factor receptor TNFRSF10B −2.07 1.53 −1.81−710.49 superfamily, member 10b NM_000633 B-cell CLL/lymphoma 2 BCL2−2.98 −1.63 −2.82 −11.36 NM_001242 CD27 molecule CD27 −3.40 −2.38 −1.35−12.72 NM_014430 Cell death-inducing CIDEB −3.48 1.56 −3.69 −2.59DFFA-like effector b NM_001065 Tumor necrosis factor receptor TNFRSF1A−4.53 2.28 −3.30 1.22 superfamily, member 1A NM_005427 Tumor protein p73TP73 −4.66 −9.80 −8.71 −26.96 NM_003844 Tumor necrosis factor receptorTNFRSF10A −4.84 −5.26 −4.33 −11.84 superfamily, member 10a NM_138578BCL2-like 1 BCL2L1 −4.94 −1.80 −6.17 −7.04 NM_001165 Baculoviral IAPBIRC3 −13.68 −1.98 −2.42 −3.42 repeat-containing 3

There is a consistent linkage to the tumor necrosis factor receptorfamily of proteins being modulated.

A strong down regulation of tumor protein p73 is also noted. Analyses ofmany tumors typically found in humans including breast and ovariancancer show a high expression of p73 when compared to normal tissues incorresponding areas. Recent finding are suggesting that deregulated overexpression of transcription factors within the body involved in cellcycle regulation and synthesis of DNA in mammalian cells (i.e.: E2F-1),induces the expression of p73. The suggestion is that p73 may be anoncoprotein, but may involve different mechanism that the related p53protein

Experiment 2 Real-time PCR Arrays using Oxidative Stress and AntioxidantDefense Array

To identify proteins that were involved in the Q10 response, real-timepolymerase chain reaction (RT-PCR) methods were employed to identifychanges in the level of mRNA's for genes/proteins involved in targetedpathway arrays for oxidative stress and antioxidant defense.

Table 10 below lists the genes that are regulated in SK-MEL28 cells with100 μM Q10 treatment. Results are given only for those genes that areregulated in two independent experiments. Although there is asignificant amount of gene regulation seen at 6 hours, most significantchanges in RNA levels are seen at 48 hours.

TABLE 10 Genes in SK-MEL-28 cells that are regulated by 100 μM Q10treatement as seen in the Oxidative Stress and Antioxidant DefenseArrays. Symbo1 Description Regulation Location Possible Functions. ALBAlbumin Down Regulation at Extracellular Carrier protein, anti-apoptotic48 hours space AOX1 Aldehyde oxidase 1 Up regulation from CytoplasmProduces free radicals, drug 16 hours metabolic process. APOEApolipoprotein E Down Regulation at Extracellular Lipid metabolism 48hours space ATOX1 ATX1 antioxidant protein 1 Down Regulation atCytoplasm Copper metabolism homolog (yeast) 48 hours BNIP3BCL2/adenovirus E1B Down Regulation at Cytoplasm Anti-apoptotic 19 kDainteracting protein 3 48 hours CSDE1 Cold shock domain Down Regulationat Cytoplasm Transcriptional regulation. containing E1, RNA- 48 hoursbinding CYBA Cytochrome b-245, alpha Down Regulation at CytoplasmApoptotic, polypeptide 48 hours CYGB Cytoglobin Down Regulation atCytoplasm Peroxidase, Transporter. 48 hours DHCR24 24-dehydrocholesterolDown Regulation at Cytoplasm Electron carrier, binds to reductase 6hours TP53, involved in apoptosis. DUOX1 Dual oxidase 1 Up Regulation at48 Plasma Calcium ion binding, electron hours Membrane carrier. DUOX2Dual oxidase 2 Down Regulation at Unknown Calcium ion binding. 48 hoursEPHX2 Epoxide hydrolase 2, Down Regulation at Cytoplasm Arachidonicacide cytoplasmic 48 hours metabolism. EPX Eosinophil peroxidase DownRegulation at Cytoplasm Phenyl alanine metabolism, 48 hours apoptosis.GPX2 Glutathione peroxidase 2 Down Regulation at Cytoplasm Electroncarrier, binds to (gastrointestinal) 48 hours TP53, involved inapoptosis. GPX3 Glutathione peroxidase 3 Up Regulation at 48Extracellular Arachidonic acid metabolims, (plasma) hours space upregulated in carcinomas. GPX5 Glutathione peroxidase 5 Up Regulation at48 Extracellular Arachidonic acid metabolism. (epididymal androgen-hours space related protein) GPX6 Glutathione peroxidase 6 DownRegulation at Extracellular Arachidonic acid metabolism. (olfactory) 48hours space GSR Glutathione reductase Down Regulation at CytoplasmGlutamate and glutathione 48 hours metabolism, apoptosis. GTF2I Generaltranscription factor Down Regulation at Nucleus Transcriptionalactivator, II, i 6 hours transcription of fos. KRT1 Keratin 1(epidermolytic Up Regulation at 48 Cytoplasm Sugar Binding.hyperkeratosis) hours LPO Lactoperoxidase Down Regulation atExtracellular Phenyl alanine metabolism. 48 hours space MBL2Mannose-binding lectin Down Regulation at Extracellular Complementsignaling, (protein C) 2, soluble 48 hours space pattern recognition in(opsonic defect) receptors. MGST3 Microsomal glutathione S- Upregulationat 16 Cytoplasm Xenobiotic metabolism. transferase 3 hours MPOMyeloperoxidase Down Regulation at Cytoplasm Anti-apoptotic, phenyl 48hours alanine metabolism. MPV17 MpV17 mitochondrial Down Regulation atCytoplasm Maintenance of inner membrane protein 6 hours mitochondrialDNA. MT3 Metallothionein 3 Down Regulation at Cytoplasm Copper ionbinding. 48 hours NCF1 Neutrophil cytosolic factor Down RegulationCyoplasm Produces free radicals. 1, (chronic granulomatous from 6 hoursdisease, autosomal 1) NCF2 Neutrophil cytosolic factor Up Regulation at48 Cytoplasm Electron carrier. 2 (65 kDa, chronic hours granulomatousdisease, autosomal 2) NME5 Non-metastatic cells 5, Down Regulation atUnknown Kinase, Purine and protein expressed in 48 hours pyrimidinemetabolism. (nucleoside-diphosphate kinase) NOS2A Nitric oxide synthase2A Down Regulation at Cytoplasm Glucocorticoid receptor (inducible,hepatocytes) 48 hours signaling, apoptosis. OXR1 Oxidation resistance 1Down Regulation at Cytoplasm Responds to oxidative stress. 48 hoursPDLIM1 PDZ and LIM domain 1 Up Regulation at 48 CytoplasmTranscriptional activator. (elfin) hours PIP3-E Phosphoinositide-bindingDown Regulation at Cytoplasm Peroxidase. protein PIP3-E 48 hours PRDX2Peroxiredoxin 2 Down Regulation at Cytoplasm Role in phenyl alanine 6hours metabolism. Role in cell death. PRDX4 Peroxiredoxin 4 DownRegulation Cytoplasm Thioredoxin peroxidase. from 24 hours PREX1Phosphatidylinositol 3,4,5- Down Regulation at Cytoplasm Forms oxygenfree radicals. trisphosphate-dependent 48 hours RAC exchanger 1 PRG3Proteoglycan 3 Down Regulation at Extracellular Role in cell death. 48hours space PTGS1 Prostaglandin- Down Regulation at Cytoplasmarachidonic acid metabolism, endoperoxide synthase 1 48 hoursprostaglandin synthesis. (prostaglandin G/H synthase and cyclooxygenase)PTGS2 Prostaglandin- Up Regulation at 48 Cytoplasm arachidonic acidmetabolism, endoperoxide synthase 2 hours prostaglandin synthesis.(prostaglandin G/H synthase and cyclooxygenase) PXDN Peroxidasin homologUp Regulation at 48 Unknown binds to TRAF4, calcium ion (Drosophila)hours binding, iron ion binding. PXDNL Peroxidasin homolog DownRegulation at Unknown peroxidase, calcium ion (Drosophila)-like 48 hoursbinding, iron ion binding. RNF7 Ring finger protein 7 Up Regulation at16 Nucleus apoptotic, copper ion binding, hours ubiquitin pathway. SGK2Serum/glucocorticoid Down Regulation at Cytoplasm Kinase, potasiumchannel regulated kinase 2 48 hours regulator. SIRT2 Sirtuin (silentmating type Up regulation at 16 Nucleus Transcription factor.information regulation 2 hours homolog) 2 (S. cerevisiae) SOD1Superoxide dismutase 1, Up Regulation at 16 Cytoplasm Apoptotic, CaspaseActivator. soluble (amyotrophic hours lateral sclerosis 1 (adult)) SOD2Superoxide dismutase 2, Up regulation at 16 Cytoplasm Apoptotic,Regulated by mitochondrial hours TNF. SOD3 Superoxide dismutase 3, DownRegulation at Extracellular Pro-apoptotic extracellular 48 hours spaceSRXN1 Sulfiredoxin 1 homolog (S. Down Regulation at Cytoplasm DNAbinding, oxidoreductase cerevisiae) 48 hours TPO Thyroid peroxidase DownRegulation at Plasma iodination of thyroglobulin, 48 hours Membranetyrosine metabolism, phenylalanine metabolism. TTN Titin Down Regulationat Cytoplasm Actin cytoskeleton signaling, 48 hours integrin signalingTXNDC2 Thioredoxin domain- Down Regulation at Cytoplasm Pyrimidinemetabolism containing 2 (spermatozoa) 48 hours

The Neutrophil cytosolic factor 2 (NCF2, 65 kDa, chronic granulomatousdisease, autosomal 2) was one of the initial top induced mRNA's(observed at 6 hours). Subsequently at the 16 hour time point andonward, Neutrophil cytosolic factor 1 (NCF1) (chronic granulomatousdisease, autosomal 1) was induced at very high levels after an initiallag phase.

Neutrophil cytosolic factor 2 is the cytosolic subunit of themulti-protein complex known as NADPH oxidase commonly found inneutrophils. This oxidase produces a burst of superoxide which isdelivered to the lumen of the neutrophil phagosome.

The NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase)is a membrane-bound enzyme complex. It can be found in the plasmamembrane as well as in the membrane of phagosome. It is made up of sixsubunits. These subunits are: a Rho guanosine triphosphatase (GTPase),usually Rac1 or Rac2 (Rac stands for Rho-related C3 botulinum toxinsubstrate)

-   -   Five “phox” units. (Phox stands for phagocytic oxidase.)        -   P91-PHOX (contains heme)        -   p22phox        -   p40phox        -   p47phox (NCF1)        -   p67phox (NCF2)

It is noted that another NADPH oxidase levels do not change. The enzymeis NOX5, which is a novel NADPH oxidase that generates superoxide andfunctions as a H+ channel in a Ca(2+)-dependent manner

In addition Phosphatidylinositol 3,4,5-trisphosphate-dependent RACexchanger 1(PREX1) was also upregulated. This protein acts as a guaninenucleotide exchange factor for the RHO family of small GTP-bindingproteins (RACs). It has been shown to bind to and activate RAC1 byexchanging bound GDP for free GTP. The encoded protein, which is foundmainly in the cytoplasm, is activated byphosphatidylinositol-3,4,5-trisphosphate and the beta-gamma subunits ofheterotrimeric G proteins.

The second major early induced protein was Nitric oxide synthase 2A(inducible, hepatocytes) (NOS2A). Nitric oxide is a reactive freeradical which acts as a biologic mediator in several processes,including neurotransmission and antimicrobial and antitumoralactivities. This gene encodes a nitric oxide synthase which is expressedin liver and is inducible by a combination of lipopolysaccharide andcertain cytokines.

Superoxide dismutase 2, mitochondrial (SOD2) is a member of theiron/manganese superoxide dismutase family. It encodes a mitochondrialprotein that forms a homotetramer and binds one manganese ion persubunit. This protein binds to the superoxide byproducts of oxidativephosphorylation and converts them to hydrogen peroxide and diatomicoxygen. Mutations in this gene have been associated with idiopathiccardiomyopathy (IDC), premature aging, sporadic motor neuron disease,and cancer.

An example of a down regulated protein is Forkhead box M1 (FOXM1), whichis known to play a key role in cell cycle progression where endogenousFOXM1 expression peaks at S and G2/M phases. Recent studies have shownthat FOXM1, regulates expression of a large array of G2/M-specificgenes, such as Plk1, cyclin B2, Nek2 and CENPF, and plays an importantrole in maintenance of chromosomal segregation and genomic stability.The FOXM1 gene is now known as a human proto-oncogene. Abnormalupregulation of FOXM1 is involved in the oncogenesis of basal cellcarcinoma (BCC). FOXM1 upregulation was subsequently found in themajority of solid human cancers including liver, breast, lung, prostate,cervix of uterus, colon, pancreas, and brain. Further studies with BCCand Q10 should evaluate FOXM1 levels.

SKMEL-28 Cells

Further experiments were carried out using SKMEL-28 cells. The level ofmRNA present in SKMEL-28 cells treated with 100 μM Q10 were compared tothe levels in untreated cells at various time points using real-time PCRmethods (RT-PCR). The PCR array (SABiosciences) is a set of optimizedreal-time PCR primer assays on 96-well plates for pathway or diseasefocused genes as well as appropriate RNA quality controls. The PCR arrayperforms gene expression analysis with real-time PCR sensitivity and themulti-gene profiling capability of a microarray.

TABLE 11 Listing and classification of mRNA levels evaluated in theOxidative Stress and Antioxidant Defense PCR Array. After six hours oftreatment with 100 μM Q10 on SKMEL-28 cells, the largest changes to themRNA levels are indicated by highlighting the protein code (increased -bold; decreased - underlined; or no change - grey).

TABLE 12 Time course evaluation of 100 μM treatment of SKMEL-28. 6 hr 16hr 24 hr 48 hr 72 hr Refseq Symbol Description Q10 Q10 Q10 Q10 Q10NM_000265 NCF1 Neutrophil cytosolic factor 1, 0 high 3.3829 15.783831.5369 (chronic granulomatous disease, autosomal 1) NM_012423 RPL13ARibosomal protein L13a −0.9025 3.1857 2.5492 4.9253 7.82 NM_020820 PREX1Phosphatidylinositol −3.2971 2.867 0.3222 6.3719 7.4763,4,5-trisphosphate-dependent RAC exchanger 1 NM_012237 SIRT2 Sirtuin(silent mating type −0.9025 4.0829 4.4766 5.7166 6.6257 informationregulation 2 homolog) 2 (S. cerevisiae) NM_005125 CCS Copper chaperonefor −0.6206 3.0077 3.452 2.9801 6.1539 superoxide dismutase NM_181652PRDX5 Peroxiredoxin 5 −2.995 3.0454 3.5381 4.7955 6.0169 NM_016276 SGK2Serum/glucocorticoid regulated 0 0 0 0.5995 5.937 kinase 2 NM_003551NME5 Non-metastatic cells 5, protein −0.6652 3.1138 3.3694 3.1549 5.782expressed in (nucleoside- diphosphate kinase) NM_004417 DUSP1 Dualspecificity phosphatase 1 −0.6998 0.5902 2.7713 3.321 5.5375 NM_001752CAT Catalase −0.8589 2.8424 0.1046 3.8557 5.3988 NM_000041 APOEApolipoprotein E −0.8212 3.2069 −0.9543 3.7694 5.3315 NM_000101 CYBACytochrome b-245, alpha polypeptide −0.3945 4.3475 3.9208 6.2452 5.0762NM_000433 NCF2 Neutrophil cytosolic factor 2 1.2266 3.0077 0.0954 5.4760 (65 kDa, chronic granulomatous disease, autosomal 2) NM_000963 PTGS2Prostaglandin-endoperoxide −0.6912 2.7046 2.6552 4.0553 −3.3022 synthase2 (prostaglandin G/H synthase and cyclooxygenase) NM_183079 PRNP Prionprotein (p27-30) −0.2144 3.5236 2.9086 5.0837 −3.9396 (Creutzfeldt-Jakobdisease, Gerstmann-Strausler-Scheinker syndrome, fatal familialinsomnia) NM_004052 BNIP3 BCL2/adenovirus E1B 19 kDa −2.9376 3.32884.312 −18.2069 −4.8424 interacting protein 3 NM_000242 MBL2Mannose-binding lectin (protein C) −0.3622 −1.9072 −3.0142 −1.1854−6.4544 2, soluble (opsonic defect) NM_021953 FOXM1 Forkhead box M1−0.8135 0.068 −0.9216 3.3655 −10.0953 The mRNA level changes weremonitored by RT-PCR methods and oxidative stress and antioxidant defenseproteins array was evaluated.

The Neutrophil cytosolic factor 2 (NCF2, 65 kDa, chronic granulomatousdisease, autosomal 2) was one of the initial top induced mRNA's(observed at 6 hours). Subsequently at the 16 hour time point andonward, Neutrophil cytosolic factor 1 (NCF1) (chronic granulomatousdisease, autosomal 1) was induced at very high levels after an initiallag phase.

Neutrophil cytosolic factor 2 is the cytosolic subunit of themulti-protein complex known as NADPH oxidase commonly found inneutrophils. This oxidase produces a burst of superoxide which isdelivered to the lumen of the neutrophil phagosome. The NADPH oxidase(nicotinamide adenine dinucleotide phosphate-oxidase) is amembrane-bound enzyme complex. It can be found in the plasma membrane aswell as in the membrane of phagosome. It is made up of six subunits.These subunits are:

a Rho guanosine triphosphatase (GTPase), usually Rac1 or Rac2 (Racstands for Rho-related C3 botulinum toxin substrate)

Five “phox” (phagocytic oxidase) units.

-   -   P91-PHOX (contains heme)    -   p22phox    -   p40phox    -   p47phox (NCF1)    -   p67phox (NCF2)

It is noted that another NADPH oxidase levels do not change. The enzymeis NOX5, which is a novel NADPH oxidase that generates superoxide andfunctions as a H+ channel in a Ca(2+)-dependent manner

In addition Phosphatidylinositol 3,4,5-trisphosphate-dependent RACexchanger 1(PREX1) was also upregulated. This protein acts as a guaninenucleotide exchange factor for the RHO family of small GTP-bindingproteins (RACs). It has been shown to bind to and activate RAC1 byexchanging bound GDP for free GTP. The encoded protein, which is foundmainly in the cytoplasm, is activated byphosphatidylinositol-3,4,5-trisphosphate and the beta-gamma subunits ofheterotrimeric G proteins.

The second major early induced protein was Nitric oxide synthase 2A(inducible, hepatocytes) (NOS2A). Nitric oxide is a reactive freeradical which acts as a biologic mediator in several processes,including neurotransmission and antimicrobial and antitumoralactivities. This gene encodes a nitric oxide synthase which is expressedin liver and is inducible by a combination of lipopolysaccharide andcertain cytokines.

An example of a down regulated protein is FOXM1, which is known to playa key role in cell cycle progression where endogenous FOXM1 expressionpeaks at S and G2/M phases. Recent studies have shown that FOXM1,regulates expression of a large array of G2/M-specific genes, such asPlk1, cyclin B2, Nek2 and CENPF, and plays an important role inmaintenance of chromosomal segregation and genomic stability. The FOXM1gene is now known as a human proto-oncogene. Abnormal upregulation ofFOXM1 is involved in the oncogenesis of basal cell carcinoma (BCC).FOXM1 upregulation was subsequently found in the majority of solid humancancers including liver, breast, lung, prostate, cervix, uterus, colon,pancreas, and brain.

Experiment 3 Real-Time PCR Arrays Using Heat Shock Array

Heat Shock Arrays were run for SCC cells and the data of regulated genesis summarized below in Table 13.

TABLE 13 Genes from the Heat Shock Protein array regulated with 100 μMQ10 treatment in SCC cells. Symbol Description Regulation. Location.Possible functions. CCT6B Chaperonin Down regulated Cytoplasm Proteinfolding and containing TCP1, at 24 hours protein complex subunit 6B(zeta 2) assembly. DNAJA1 DnaJ (Hsp40) Up regulated at 6 NucleusResponds to DNA homolog, subfamily hours. damage and changes in A,member 1 protein folding. DNAJB13 DnaJ (Hsp40) Down regulated UnknownProtein folding and related, subfamily B at 6 hours. apoptosis. member13 DNAJB5 DnaJ (Hsp40) Down regulated Unknown Binds to HSP, involved inhomolog, subfamily at 6 hours. protein folding and in B, member 5protein complex assembly. DNAJC12 DnaJ (Hsp40) Down regulated UnknownBinds to HSP, involved in homolog, subfamily at 6 hours. protein foldingand in C, member 12 protein complex assembly. DNAJC4 DnaJ (Hsp40) Downregulated Cytoplasm Binds to HSP, involved in homolog, subfamily at 6hours. protein folding and in C, member 4 protein complex assembly.DNAJC5B DnaJ (Hsp40) Down regulated Unknown Involved in protein homolog,subfamily at 6 hours. folding responds to C, member 5 beta changes inprotein folding. HSPA8 Heat shock 70 kDa Up regulated at 6 CytoplasmRegulates TNF, binds protein 8 hours. BAG1, STUB1, TP53, involved inapoptosis. HSPH1 Heat shock Up regulated at 6 Cytoplasm Binds to HSPA8,1051 kDa/1101 kDa hours. important for protein protein 1 folding,responds to protein unfolding and stress.

Experiment 4 Real-Time PCR Arrays Using Diabetes Array

The experiments described in this example were performed to test theoverall hypothesis that Q10 would have an impact on multiple genes andalter the metabolic state of a cell. The mRNA from SKMEL-28 cellstreated with 100 μM Q10 was evaluated by RT-PCR against a panel oftarget proteins involved in diabetes and related pathways. Results fromthis experiment demonstrate that several proteins involved in glycolyicpathways and insulin processing are altered in their mRNA expressionlevels (summarized in Table 14).

TABLE 14 Major mRNA level changes to SKMEL-28 cells treated with 100 μMQ10 for 16 hours. Fold Change after 16 hours Refseq Description Symbol(100 μM Q10) NM_000162 Glucokinase GCK 8.5386 (hexokinase 4) NM_178849Hepatocyte nuclear HNF4A 8.421 factor 4, alpha NM_005249 Forkhead box G1FOXG1 4.6396 NM_000599 Insulin-like growth factor IGFBP5 2.2721 bindingprotein 5 NM_001101 Actin, beta ACTB −2.0936 NM_002863 Phosphorylase,glycogen; PYGL −2.65 liver (Hers disease, glycogen storage disease typeVI) NM_001065 Tumor necrosis factor TNFRSF1A −2.8011 receptor super-family, member 1A NM_021158 Tribbles homolog 3 TRIB3 −2.8011(Drosophila) NM_003749 Insulin receptor IRS2 −2.9404 substrate 2NM_004578 RAB4A, member RAS RAB4A −3.1296 oncogene family NM_004176Sterol regulatory element SREBF1 −3.5455 binding tran- scription factor1 NM_004969 Insulin-degrading IDE −4.4878 enzyme NM_005026Phosphoinositide- PIK3CD −6.8971 3-kinase, catalytic, delta polypeptideNM_000208 Insulin receptor INSR −8.6099 NM_003376 Vascular endothelialVEGFA −15.5194 growth factor A NM_001315 Mitogen-activated MAPK14−74.3366 protein kinase 14

The results of this initial experiment show that the mRNA levels for avariety of insulin related proteins were modulated in both directions.The results indicate that Q10 would have an impact on diabetic diseasetreatment and/or evaluation.

Further experiments were next conducted to confirm the results aboveobtained from SK-MEL-28 cells treated with Q10. Many of the genes inSK-MEL-28 cells are regulated as early as 6 hours after Q10 treatment.However, the initial regulation becomes less evident by 16 and 24 hours.Around 48 hours, we find that many of the genes in the Diabetes arrayare again strongly regulated. Results that are consistent from two ormore or independent experiments are summarized below in Table 15. SCCcells also appeared to exhibit regulation in some genes, both at 6 and24 hours after Q10 treatment. These results from SCC cells aresummarized in Table 16 while genes that are regulated both in SK-MEL-28cells and in SCC cells are summarized in Table 17.

TABLE 15 Genes in SK-MEL-28 cells regulated by 100 μM Q10 treatment whenanalyzed by the Diabetes Array. Symbol Description Regulation. LocationPossible Function ADRB3 Adrenergic, beta-3-, Down Regulated Plasma cAMPsignaling, G- receptor at 48 hours membrane protein signaling CEACAM1Carcinoembryonic Down Regulated Extracellular Anti-apoptotic,antigen-related cell at 48 hours space positive regulation of adhesionmolecule 1 angiogenesis. (biliary glycoprotein) CEBPA CCAAT/enhancer Upregulated at Nucleus Glucocorticoid binding protein (C/EBP), 48 hoursreceptor signaling, alpha VDR/RXR activation. CTLA4 CytotoxicT-lymphocyte- Down Regulated Plasma T cell receptor associated protein 4at 48 hours Membrane signaling, activates CASP8. DUSP4 Dual specificityDown Regulated Nucleus Phosphatase phosphatase 4 at 48 hours ENPP1Ectonucleotide Down Regulated Plasma Negative regulator pyrophosphatase/at 48 hours membrane of the insulin phosphodiesterase 1 receptor pathwayFOXC2 Forkhead box C2 (MFH- Down Regulated Nucleus Anti-apoptotic, 1,mesenchyme at 48 hours transcription factor forkhead 1) G6PDGlucose-6-phosphate Up regulated at Cytoplasm Pentose Phosphatedehydrogenase 48 hours, then Pathway, down regulated Glutathionemetabolism. HMOX1 Heme oxygenase Down Regulated Cytoplasm Heme oxygenase(decycling) 1 at 48 hours decycling ICAM1 Intercellular adhesion DownRegulated Plasma Regulated by molecule 1 (CD54), at 48 hours membraneatorvastatin, human rhinovirus processes some receptor caspases. IL4RInterleukin 4 receptor Down Regulated Plasma Up regulation by at 48hours membrane TP73, binds to IRS1 and IRS2 IRS1 Insulin receptor Upregulated at Plasma Binds Insulin substrate 1 48 hours then membranereceptor down regulated IRS2 Insulin receptor Down Regulated PlasmaIGF-1 signaling substrate 2 at 48 hours membrane NSF N-ethylmaleimide-Down Regulated Cytoplasm GABA signaling sensitive factor at 48 hoursPIK3CD Phosphoinositide-3- Down Regulated Cytoplasm Kinase kinase,catalytic, delta at 48 hours polypeptide PPARG Peroxisome proliferator-Down Regulated Nucleus Transcriptional activated receptor at 48 hoursfactor gamma PRKCB1 Protein kinase C, beta 1 Down Regulated CytoplasmPKC family at 48 hours SELL Selectin L (lymphocyte Down Regulated PlasmaActivates RAS, adhesion molecule 1) at 48 hours membrane MAPK SREBF1Sterol regulatory Up regulated at Nucleus Transcriptional elementbinding 48 hours then factor transcription factor 1 down regulatedSTXBP1 Syntaxin binding protein Down Regulated Cytoplasm Present inmyelin 1 at 48 hours enriched fraction. TGFB1 Transforming growth Upregulated at Extracellular Pro-apoptotic factor, beta 1 48 hours thenspace down regulated NKX2-1 NK2 homeobox 1 Down Regulated NucleusTranscriptional at 48 hours activator TNF Tumor necrosis factor Upregulated at Extracellular Pro-apoptotic (TNF superfamily, 48 hoursspace member 2) TNFRSF1A Tumor necrosis factor Down Regulated PlasmaPro-apoptotic receptor superfamily, at 72 hours membrane member 1A VEGFAVascular endothelial Up regulated at Cytoplasm Kinase growth factor A 58hours then down regulated

TABLE 16 Genes in SCC cells regulated by 100 μM Q10 treatment whenanalyzed by the Diabetes Array. Symbol Description Regulation. G6PDGlucose-6-phosphate dehydrogenase Down regulated at 6 hours. ICAM1Intercellular adhesion molecule 1 Down regulated (CD54), humanrhinovirus receptor at 6 hours. INPPL1 Inositol polyphosphate Downregulated phosphatase-like 1 at 6 hours. NOS3 Nitric oxide synthase 3Down regulated (endothelial cell) at 6 hours. PIK3CDPhosphoinositide-3-kinase, Down regulated catalytic, delta polypeptideat 6 hours. PPARA Peroxisome proliferative activated Down regulatedreceptor, alpha at 6 hours. PYGL Phosphorylase, glycogen; liver Downregulated (Hers disease, glycogen storage at 6 hours. disease type VI)SREBF1 Sterol regulatory element binding Down regulated transcriptionfactor 1 at 6 hours. STXBP2 Syntaxin binding protein 2 Down regulated at6 hours. TNF Tumor necrosis factor (TNF Down regulated superfamily,member 2) at 6 hours. TNFRSF1A Tumor necrosis factor receptor Downregulated superfamily, member 1A at 6 and 24 hours. VEGFA Vascularendothelial Down regulated growth factor A at 6 hours.

TABLE 17 Genes from the diabetes array regulated with 100 μM Q10treatment for both SK-MEL-28 and SCC cells. Symbol Description. G6PDGlucose-6-phosphate dehydrogenase ICAM1 Intercellular adhesion molecule1 (CD54), human rhinovirus receptor PIK3CD Phosphoinositide-3-kinase,catalytic, delta polypeptide SREBF1 Sterol regulatory element bindingtranscription factor 1 TNF Tumor necrosis factor (TNF superfamily,member 2) TNFRSF1A Tumor necrosis factor receptor superfamily, member 1AVEGFA Vascular endothelial growth factor A

The mRNA levels for a variety of insulin related proteins were modulatedin both directions. Q10 has an impact on regulation of cellularmetabolism, and thus influences metabolic disregluation diseases such asdiabetes. Two proteins that were significantly modulated are furtherdiscussed below.

Mitogen-activated protein kinase 14 (MAPK14): Mitogen-activated proteinkinase 14 (MAPK14) is a member of the MAP kinase family. MAP kinases actas an integration point for multiple biochemical signals, and areinvolved in a wide variety of cellular processes such as proliferation,differentiation, transcription regulation and development. Results fromthis experiment show that the MAPK14 was significantly down-regulated.

Hepatocyte nuclear factor 4, alpha (HNF4A): HNF4 (Hepatocyte NuclearFactor 4) is a nuclear receptor protein mostly expressed in the liver,gut, kidney, and pancreatic beta cells that is critical for liverdevelopment. In humans, there are two isoforms of NHF4, alpha and gammaencoded by two separate genes HNF4A and HNF4G respectively. (See, e.g.,Chartier F L, Bossu J P, Laudet V, Fruchart J C, Laine B (1994).“Cloning and sequencing of cDNAs encoding the human hepatocyte nuclearfactor 4 indicate the presence of two isoforms in human liver”. Gene 147(2): 269-72.)

HNF4 was originally classified as an orphan receptor. However HNF4 wasfound later to be constitutively active by virtue of being continuouslybound to a variety of fatty acids. (See, e.g., Sladek F (2002).“Desperately seeking . . . something”. Mol Cell 10 (2): 219-221 and JumpD B, Botolin D, Wang Y, Xu J, Christian B, Demeure I (2005). “Fatty acidregulation of hepatic gene transcription”. J Nutr 135 (11)). The ligandbinding domain of HNF4, as with other nuclear receptors, adopts acanonical alpha helical sandwich fold (see, e.g., Wisely G B, Miller AB, Davis R G, Thornquest A D Jr, Johnson R, Spitzer T, Sefler A, ShearerB, Moore J T, Miller A B, Willson T M, Williams S P (2002). “Hepatocytenuclear factor 4 is a transcription factor that constitutively bindsfatty acids”. Structure 10 (9): 1225-34 and Dhe-Paganon S, Duda K,Iwamoto M, Chi Y I, Shoelson S E (2002). “Crystal structure of the HNF4alpha ligand binding domain in complex with endogenous fatty acidligand”. J Biol Chem 277 (41): 37973-6) and interacts with co-activatorproteins. (See, e.g., Duda K, Chi Y I, Shoelson S E (2004). “Structuralbasis for HNF-4-alpha activation by ligand and coactivator binding”. JBiol Chem 279 (22): 23311-6). Mutations in the HNF4-α gene have beenlinked to maturity onset diabetes of the young (MODY). (See, e.g.,Fajans S S, Bell G I, Polonsky K S (2001). “Molecular mechanisms andclinical pathophysiology of maturity-onset diabetes of the young”. NEngl J Med 345 (13): 971-80.)

Hepatocyte nuclear factor 4 (HNF4) is a tissue-specific transcriptionfactor known to regulate a large number of genes in hepatocytes andpancreatic cells. Although HNF4 is highly expressed in some sections ofthe kidney, little is known about its role in this organ and aboutHNF4-regulated genes in the kidney cells. The abundance and activity ofHNF4 are frequently reduced in renal cell carcinoma (RCC) indicatingsome tumor suppressing function of HNF4 in renal cells. Interestingly,many of the genes regulated by HNF4 have been shown to be deregulated inRCC microarray studies. These genes (ACY1, WT1, SELENBP1, COBL, EFHD1,AGXT2L1, ALDH5A1, THEM2, ABCB1, FLJ14146, CSPG2, TRIM9 and HEY1) aregood candidates for genes whose activity is changed upon the decrease ofHNF4 in RCC.

In the structure of the ligand binding domain of HNF4alpha (1M7W.pdb;Dhe-Paganon (2002) JBC, 277, 37973); a small lipid was observed andwhich co-purified from E. coli production. The crystal contains twoconformations of the protein, where the elongated helix 10 and shorthelix 12 have alternate conformations. Upon examination of the lipidbinding region, it was interesting to observe that there are two exitsregions. One exit region holds the small lipids head group, and it isnoted that several pocket regions are co-localized with this exit port.A hypothesis would be that Q10 binds specifically to this transcriptionfactor. When Q10 in modeled into this lipid binding tunnel, the Q10 ringwould fit into the surface pocket (FIG. 28). A known loss-of-functionmutation (E276Q) would have the potential to order the residues liningthis surface pocket, and thus have a negative impact on the putative Q10binding.

In addition, with this Q10 binding model, the hydrophobic tail wouldextend out of the internal cavity and would then interact with theelongated helix 10. Thus, this interaction could potential alter theconformation of the helix 10/12 group. This may then alter theactivation/inactivation equilibrium of the transcription factoractivity.

Example 7 Antibody MicroArray Analysis

The evaluation of protein concentration due to the presence of Q10 wasevaluated through the utilization of antibody microarray methods. Themicroarray contained antibodies for over 700 proteins, sampling a broadrange of protein types and potential pathway markers.

An initial experiment to assess changes at the protein concentrationlevel in cells treated with Q10 was conducted with an antibodymicroarray (Panorama XP725 Antibody Array, Sigma) and SK-MEL-28 cellstreated for 6 or 24 hour. The cells were harvested and extracted toobtain a soluble protein supernatant. Two portions of protein (˜1 mgtotal) from each sample (at 1 mg/mL) were each label with fluorescentdye (Cy3 and Cy5, respectively). The excess dye was removed from theprotein and the material utilized for the microarray incubations. Tocompare two time point samples, equal amounts of protein were mixed,with each sample being of the different label type (e.g., 3 hour extractlabeled with Cy3 was mixed with the 24 hour extract labeled with Cy5).After incubation with the microarray chip (according to manufacturesrecommended protocols), the chips were washed and dried. The microarrayswere scanned with a fluorescent laser scanner to measure the relativefluorescence intensity of the Cy3 and Cy5 dyes.

TABLE 18 Proteins with increased levels in SK-MEL-28 cells after 24 hourtreatment with 50 μM Q10 Name Ratio Cdk1 0.1 DcR1 0.1 Protein Kinase Cb20.1 Tumor Necrosis Factor 0.1 Soluble Receptor II BAD 0.1 Caspase13 0.2FBI1 PAKEMON 0.2 Zyxin 0.2 Cdc25A 0.3 PIASx 0.3 Nerve Growth Factor b0.3 Protein Tyrosine 0.3 Phosphatase PEST hBRM hSNF2a 0.4 GRP94 0.4Calmodulin 0.4 Serine Threonine Protein 0.4 Phosphatase 2C a b ARC 0.4NeurabinII 0.4 Nitric Oxide 0.4 Synthase bNOS Serine Threonine Protein0.4 Phosphatase 1b Heat Shock Protein 110 0.4 Serine Threonine Protein0.4 Phosphatase 1g1 COX II 0.5 HSP70 0.5 BLK 0.5 Cytokeratin 8 12 0.5BUBR1 0.5 FOXC2 0.5 Serine Threonine Protein 0.5 Phosphatase 2 A Bg MSH60.5 DR6 0.5 Rad17 0.5 BAF57 0.5 Transforming Growth 0.5 Factorb pan BTK0.5 SerineThreonine Protein 0.5 Phosphatase 2 A/B pan2 CNPase 0.5 SynCAM0.5 Proliferating Cell Nuclear 0.5 Antigen

TABLE 19 Proteins with increased levels in SK-MEL-28 cells after 24 hourtreatment with 50 μM Q10 Name Ratio BclxL 4.2 BID 3.7 Bmf 3.7 PUMA bbc33.0 Zip Kinase 2.8 Bmf 2.8 DcR2 2.7 E2F1 2.7 FAK pTyr577 2.5 FKHRL1FOXO3a 2.5 MTBP 2.5 Connexin 32 2.5 Annexin VII 2.4 p63 2.4 SUMO1 2.4IAfadin 2.3 MDMX 2.3 Pyk2 2.3 RIP Receptor 2.3 Interacting Protein RICK2.3 IKKa 2.3 Bclx 2.3 Afadin 2.2 Proliferating Cell 2.2 Protein Ki67Histone H3 pSer28 2.2 CASK LIN2 2.2 Centrin 2.2 TOM22 2.1 Nitric OxideSynthase 2.1 Endothelial eNOS Protein Kinase Ba 2.1 Laminin 2.1 MyosinIb Nuclear 2.1 Caspase 7 2.1 MAP Kinase 2 ERK2 2.1 KIF17 2.1 Claspin 2.1GRP75 2.1 Caspase 6 2.1 ILP2 2.1 aActinin 2.1 Vitronectin 2.1 DRAK1 2.1PTEN 2.1 Grb2 2.1 HDAC4 2.0 HDAC7 2.0 Nitric Oxide 2.0 Synthase bNOSHDAC2 2.0 p38 MAPK 2.0 Reelin 2.0 Protein Kinase Cd 2.0 cerbBS 2.0 hSNF5INI1 2.0 Protein Kinase Ca 2.0 Glutamate receptor 2.0 NMDAR 2a Leptin2.0 Dimethyl Histone H3 2.0 diMeLys4 BID 2.0 MeCP2 2.0 Nerve growthfactor 2.0 receptor p75 Myosin Light 2.0 Chain Kinase cRaf pSer621 2.0GRP78 BiP 2.0 cMyc 2.0 Raf1 2.0 MTA2 MTA1L 2.0 Sir2 2.0 ATF2 pThr69 712.0 Protein Kinase C 2.0 Protein Kinase Cb2 2.0

In order to confirm the previously observed apoptosis proteins, and toexpand the evaluation into a larger number of pro-apoptosis andanti-apoptosis proteins, two assay methods were chosen which werecapable of screening the broad family of proteins potentially involved.

First, an antibody micro array (Panorama XP725 Antibody Array, Sigma)was utilized to screen over 700 protein antibodies to assess changes atthe protein concentration level in SK-MEL-28 cells treated for 24 hourswith 50 μM Q10.

From the Antibody array experiments, on SKMEL-28 with Q10 (24 hr), thefollowing are some of the identified proteins with altered levels:Bcl-x1, Bmf, BTK, BLK, cJun (pSer63), Connexin 32, PUMA bbc3, BID, Par4,cCb1. The key conclusion from this initial study was that the expectedpro-apoptosis proteins are altered.

Antibody Microarray for SK-MEL-28

An antibody micro array (Panorama XP725 Antibody Array, Sigma) wasutilized to screen over 700 protein antibodies to assess changes at theprotein concentration level in SK-MEL-28 cells treated for 24 hours with50 μM Q10.

TABLE 20 Changes in protein levels in SKMEL-28 treated with 50 μM Q10Antibody SKMEL28 Q10/ SKMEL28/ HEKa Q10/ Number SKMEL28 HEKa HEKa Name(Sigma) control control control BclxL B9429 2.46 1.04 1.83 PUMA bbc3P4743 2.31 1.14 2.14 Bmf B1559 2.23 1.12 2.11 Bmf B1684 2.09 1.13 1.74cJun pSer63 J2128 1.99 1.14 1.85 BLK B8928 1.94 1.05 1.51

From the Antibody array experiments, on SKMEL-28 with Q10 (24 hr), thefollowing are some of the identified proteins with altered levels:Bcl-x1, Bmf, BTK, BLK, cJun (pSer63), Connexin 32, PUMA bbc3, BID, Par4,cCb1. These data confirm that the levels of pro-apoptosis proteins arealtered upon incubation with elevated levels of exogenously added Q10.

Bcl-x1 (“Basal cell lymphoma-extra large”) is a transmembrane moleculein the mitochondria. It is involved in the signal transduction pathwayof the FAS-L and is one of several anti-apoptotic proteins which aremembers of the Bcl-2 family of proteins. It has been implicated in thesurvival of cancer cells. However, it is known that alternative splicingof human Bcl-x mRNA may result in at least two distinct Bcl-x mRNAspecies, Bcl-xL and Bcl-xS. The predominant protein product (233 aminoacids) is the larger Bcl-x mRNA, Bcl-xL, which inhibits cell death upongrowth factor withdrawal (Boise et al., 1993. Cell 74, 597-608). Bcl-xS,on the other hand, inhibits the ability of Bcl-2 to inhibit cell deathand renders cells more susceptible to apoptotic cell death.

TABLE 21 Proteins with increased levels in SCC cells after 24 hourtreatment with 100 μM Q10. Name Ratio PUMA bbc3 3.81 HDAC7 3.21 BID 3.12MTBP 3.00 p38 MAP Kinase 2.93 NonActivated PKR 2.87 TRAIL 2.86 DR5 2.86Cdk3 2.82 NCadherin 2.71 Reelin 2.68 p35 Cdk5 Regulator 2.63 HDAC10 2.60RAP1 2.59 PSF 2.56 cMyc 2.55 methyl Histone H3 2.54 MeLys9 HDAC1 2.51F1A 2.48 ROCK1 2.45 Bim 2.45 FXR2 2.44 DEDAF 2.44 DcR1 2.40 APRIL 2.40PRMT1 2.36 Pyk2 pTyr580 2.34 Vitronectin 2.33 Synaptopodin 2.32Caspase13 2.30 Syntaxin 8 2.29 DR6 2.29 BLK 2.28 ROCK2 2.28 Sir2 2.25DcR3 2.24 RbAp48 RbAp46 2.21 OGlcNAc Transferase 2.21 GRP78 BiP 2.20Sin3A 2.20 p63 2.20 Presenilin1 2.19 PML 2.18 PAK1pThr212 2.17 HDAC82.16 HDAC6 2.15 Nitric Oxide Synthase 2.15 Inducible iNOS Neurofibromin2.15 Syntaxin 6 2.13 Parkin 2.12 Rad17 2.11 Nitric Oxide 2.10 SynthasebNOS TIS7 2.09 OP18 Stathmin 2.08 (stathmin 1/ oncoprotein 18)phospho-b-Catenin 2.07 pSer45 NeurabinII 2.07 e Tubulin 2.07 PKB pThr3082.07 Ornithine Decarboxylase 2.07 P53 BP1 2.06 Pyk2 2.05 HDAC5 2.05Connexin 43 2.05 a1Syntrophin 2.04 MRP1 2.04 cerbB4 2.03 SNitrosocysteine 2.03 SGK 2.02 Rab5 2.01 Ubiquitin Cterminal 2.01Hydrolase L1 Myosin Ib Nuclear 2.00 Par4 Prostate 2.00 ApoptosisResponse 4

TABLE 22 Proteins with reduced levels in SCC cells after 24 hourtreatment with 100 μM Q10. Name Ratio AP1 0.68 Centrin 0.55 CUGBP1 0.67Cystatin A 0.69 Cytokeratin CK5 0.60 Fibronectin 0.63 gParvin 0.70Growth Factor 0.63 Independence1 Nerve Growth 0.60 Factor b ProCaspase 80.72 Rab7 0.62 Rab9 0.73 Serine Threonine Protein 0.71 Phosphatase 1g1Serine Threonine Protein 0.73 Phosphatase 2 A Bg SKM1 0.70 SLIPR MAGI30.67 Spectrin a and b 0.70 Spred2 0.66 TRF1 0.74

Example 8 Western Blot Analysis

The first experiment processed and evaluated by Western blot and 2-D gelelectrophoresis was carried out on the skin cancer cell line SKMEL-28.This experimental set involved SK-MEL-28 cells treated at 3, 6, 12, and24 hours with 50 or 100 μM Q10.

A variety of cell types were evaluated by Western blot analysis againstan antibody for Bcl-xL (FIG. 14), an antibody for Vimentin (FIG. 15), aseries of antibodies for mitochondrial oxidative phosphorylationfunction (FIGS. 16-21) and against a series of antibodies related tomitochondrial membrane integrity (FIGS. 22-27). The results from theseexperiments demonstrated that several of the examined proteins wereupregulated or downregulated as a result of cell treatment with Q10.

Example 9 Diabetes Related Genes Identified as being Modulated at themRNA Level by Treatment of Pancreatic Cancer Cells (PaCa2) with 100 umQ10

Diabetes arrays were run for samples treated with 100 uM Q10 at varioustimes after treatment. Experiments were carried out essentially asdescribed above. The various genes found to be modulated upon Q10treatment are summarized in Table 23 below. The results showed that thefollowing genes are modulated by Q10 treatment: ABCC8, ACLY, ADRB3,CCL5, CEACAM1, CEBRA, FOXG1, FOXP3, G6PD, GLP1R, GPD1, HNF4A, ICAM1,IGFBP5, INPPL1, IRS2, MAPK14, ME1, NFKB1, PARP1, PIK3C2B, PIK3CD,PPARGC1B, PRKAG2, PTPN1, PYGL, SLC2A4, SNAP25, HNF1B, TNRFSF1A, TRIB3,VAPA, VEGFA, IL4R and IL6.

TABLE 23

Example 10 Angiogenesis Related Genes Identified as being Modulated atthe mRNA Level by Treatment of Pancreatic Cancer Cells (PaCa2) with 100μM Q10

Angiogenesis arrays were run for samples treated with 100 uM Q10 atvarious times after treatment. Experiments were carried out essentiallyas described above. The various genes found to be modulated upon Q10treatment are summarized in Table 24 below. The results showed that thefollowing genes are modulated by Q10 treatment: AKT1, ANGPTL4, ANGPEP,CCL2, CDH4, CXCL1, EDG1, EFNA3, EFNB2, EGF, FGF1, ID3, IL1B, 1L8, KDR,NRP1, PECAM1, PROK2, SERPINF1, SPHK1, STAB1, TGFB1, VEGFA and VEGFB.

TABLE 24

Example 11 Apoptosis Related Genes Identified as being Modulated at themRNA Level by Treatment of Pancreatic Cancer Cells (PaCa2) with 100 μMQ10

Apoptosis arrays were run for samples treated with 100 uM Q10 at varioustimes after treatment. Experiments were carried out essentially asdescribed above. The various genes found to be modulated upon Q10treatment are summarized in Table 25 below. The results showed that thefollowing genes are modulated by Q10 treatment: ABL1, AKT1, Bcl2L1,BclAF1, CASP1, CASP2, CASP6, CIDEA, FADD, LTA, TNF, TNFSF10A andTNFSF10.

TABLE 25

Example 12 PCR Diabetes Arrays on Liver Cancer (HepG2) Cells

HepG2 (liver cancer) cells were treated with either the vehicle for 24hours or 100 μM Q10 for different times. The treatment was initiated on1×105 cells per well, following the procedure utilized in the PaCa2cells (above, Examples 9-11). However, the total amount of RNA that wasextracted from these samples was lower than expected. Reversetranscription is normally done using 1 μg of total RNA (determined bymeasurement at 260 nm). The maximum volume that can be used per reversetranscription is 8 μl. Since the RNA concentration was low, the RT-PCRarray analysis using the vehicle, and Q10 treated samples from 16 hoursand 48 hours was performed using 0.44 μg of RNA. The arrays provided aninitial analysis of trends and patterns in HepG2 gene regulation with100 μM Q10 treatment, as summarized in Table 26 below. The resultsshowed that each of the genes PPARGC1A, PRKAA1 and SNAP25 weredownregulated at 16 hours following treatment (by approximately 20 fold,6 fold and 5 fold, respectively). At 48 hours following treatment,PPARGC1A and PRKAA1 had normalized or were slightly upregulated, whileSNAP25 was downregulated by approximately 2 fold.

TABLE 26 List of genes regulated in the Diabetes Arrays when HepG2 cellswere treated with 100 μM Q10. Gene Gene name Gene Function. PPARGC1Aperoxisome proliferator- Involved in cell death, activated receptorgamma, proliferation, cellular coactivator 1 alpha respiration andtransmembrane potential. PRKAA1 protein kinase, AMP- Regulates TP53 andis activated, alpha 1 involved in apoptosis, catalytic subunit regulatesglycolysis, regulates metabolic enzyme activities. SNAP25synaptosomal-associated Plays in transport, protein, 25 kDa fusion,exocytosis and release of molecules.

Example 13 PCR Angiogenesis Array on Liver Cancer (HEPG2) Cells

HepG2 (liver cancer) cells were treated with either the vehicle for 24hours or 100 μM Q10 for different times. The treatment was initiated on1×105 cells per well, following the procedure utilized in the PaCa2cells (above Examples 9-11). However, the total amount of RNA that wasextracted from these samples was lower than expected. Reversetranscription is normally done using 1 μg of total RNA (determined bymeasurement at 260 nm). The maximum volume that can be used per reversetranscription is 8 μl. Since the RNA concentration was low, the RT-PCRarray analysis using the vehicle, and Q10 treated samples from 16 hoursand 48 hours was performed using 0.44 μg of RNA. The arrays provided aninitial analysis of trends and patterns in HepG2 gene regulation with100 μM Q10 treatment, as summarized in Table 27 below. The various genesfound to be modulated upon Q10 treatment are summarized in Table 27below. The results showed that each of the genes ANGPTL3, ANGPTL4,CXCL1, CXCL3, CXCL5, ENG, MMP2 and TIMP3 were upregulated at 16 hoursfollowing treatment (by approximately 5.5, 3, 3, 3.2, 3, 3, 1 and 6.5fold, 6 fold and 5 fold, respectively, over that of control). ID3 wasdownregulated at 16 hours following Q10 treatment, by approximately 5fold over control. At 48 hours following treatment, ANGPTL3, CXCL1,CXCL3, ENG and TIMP3 were still upregulated (by approximately 3.5, 1.5,3.175, 2 and 3 fold, respectively, over control), while ANGPTL4, CXCL5,ID3 and MMP2 were downregulated by approximately 1, 1, 2 and 18 fold,respectively, over control.

TABLE 27 List of genes regulated in the Angiogenesis Arrays when HepG2cells were treated with 100 μM Q10. Gene Gene Name. Gene Function.ANGPTL3 angiopoietin-like 3 Predominantly expressed in live, role incell migration and adhesion, regulates fatty acid and glycerolmetabolism. ANGPTL4 angiopoietin-like 4 Regulated by PPARG, apoptosisinhibitor for vascular endothelial cells, role lipid and glucosemetabolism and insulin sensitivity. CXCL1 chemokine (C-X-C motif) Rolein cell proliferation and migration ligand 1 (melanoma growthstimulating activity, alpha) CXCL3 chemokine (C-X-C motif) Chemokineactivation, hepatic stellar cell ligand 3 activation, migration,proliferation. CXCL5 chemokine (C-X-C motif) Produced along with IL8when stimulated ligand 5 with IL1 or TNFA. Role in chemotaxis,migration, proliferation. ENG endoglin Binds to TGFBR and is involved inmigration, proliferation, attachment and invasion. ID3 inhibitor of DNAbinding 3, Regulates MMP2, Regulated by TGFB1, dominant negative helix-Vitamin D3, Retinoic acid, VEGFA, involved loop-helix protein inapoptosis, proliferation, differentiation, migration. MMP2 matrixmetallopeptidase 2 Hepatic stellate cell activation, HIF (gelatinase A,72 kDa signaling, binds to TIMP3, involved in gelatinase, 72 kDa type IVtumorigenesis, apoptosis, proliferation, collagenase) invasiveness,migration and chemotaxis. TIMP3 TIMP metallopeptidase Regulates MMP2,ICAM1. Regulated by inhibitor 3 TGFB, EGF, TNF, FGF and TP53. Involvedin apoptosis, cell-cell adhesion and malignancy.

Proteins known to be involved in the process of angiogenesis werecomponents in the RT-PCR array. Angiogenesis is a critical process bywhich cancer cells become malignant. Some of these proteins are alsoimplicated in diabetes.

ANGPTL3 and ANGPTL4: The literature related to ANGPTL3 connects thisprotein to the regulation of lipid metabolism. In particular, theliterature (Li, C. Curr Opin Lipidol. 2006 April; 17(2):152-6) teachesthat both angiopoietins and angiopoietin-like proteins share similardomain structures. ANGPTL3 and 4 are the only two members of thissuperfamily that inhibit lipoprotein lipase activity. However, ANGPTL3and 4 are differentially regulated at multiple levels, suggestingnon-redundant functions in vivo. ANGPTL3 and 4 are proteolyticallyprocessed into two halves and are differentially regulated by nuclearreceptors. Transgenic overexpression of ANGPTL4 as well as knockout ofANGPTL3 or 4 demonstrate that these two proteins play essential roles inlipoprotein metabolism: liver-derived ANGPTL3 inhibits lipoproteinlipase activity primarily in the fed state, while ANGPTL4 playsimportant roles in both fed and fasted states. In addition, ANGPTL4regulates the tissue-specific delivery of lipoprotein-derived fattyacids. ANGPTL4 is thus an endocrine or autocrine/paracarine inhibitor oflipoprotein lipase depending on its sites of expression.

Lipoprotein lipase is an enzyme that hydrolyzes lipids in lipoproteins,such as those found in chylomicrons and very low-density lipoproteins(VLDL), into three free fatty acids and one glycerol molecule.Lipoprotein lipase activity in a given tissue is the rate limiting stepfor the uptake of triglyceride-derived fatty acids. Imbalances in thepartitioning of fatty acids have major metabolic consequences. High-fatdiets have been shown to cause tissue-specific overexpression of LPL,which has been implicated in tissue-specific insulin resistance andconsequent development of type 2 diabetes mellitus.

The results in this Example indicate that Q10 is modulating proteinsinvolved in lipid metabolism and thus warrants further investigation ofANGPTL3/ANGPTL4 and their related pathways. For example, ANGPTL3/ANGPTL4have been implicated to play a role in the following pathways: Akt,cholesterol, fatty acid, HDL-cholesterol, HNF1A, ITGA5, ITGA5, ITGAV,ITG83, L-trilodothynonine, LIPG, LPL, Mapk, Nrth, NR1H3, PPARD, PTK2,RXRA, triacylglerol and 9-cis-retinoic acid.

Example 14 PCR Apoptosis Array on Liver Cancer (HEPG2) Cells

Apoptosis arrays were run for samples treated with 100 uM Q10 for 16 and48 hours as described above. However, the array for 48 hours was runchoosing FAM as the fluorophore instead of SYBR. Both FAM and SYBRfluoresce at the same wavelength.

The various genes found to be modulated upon Q10 treatment aresummarized in Table 28 below. The results showed that CASP9 wasupregulated at 16 hours following Q10 treatment, by approximately 61fold over control, while BAG1 and TNFRSF1A were downregulated at 16hours following treatment by approximately 6 and 4 fold, respectively,over that of control. At 48 hours following treatment, CASP9, BAG1 andTNFRSF1A were upregulated by approximately 55, 1 and 1 fold,respectively, over control.

TABLE 28 List of genes regulated in the Apoptosis Arrays when HepG2cells were treated with 100 μM Q10. Gene Gene Name Gene Function. BAG1BCL2-associated athanogene Involved with Apoptosis CASP9 caspase 9,apoptosis- Apoptosis through release related cysteine of cytochrome c.peptidase TNFRSF1A tumor necrosis factor anti-apoptosis, binds receptorsuperfamily, many cell death member 1A factors, regulates ICAM1

Example 15 Assessing Ability of Epi-Shifter to Treat Metabolic Disorder

In order to determine if a selected Epi-shifter, e.g., CoQ10, is capableof treating a metabolic disorder, e.g., diabetes, cell based assays thatmonitor an increase in insulin-stimulated glucose uptake in vitro areemployed. In particular, differentiated mouse adipocytes are used toidentify agents that have the ability to increase glucose uptake uponinsulin stimulation, as detected by scintillation counting ofradiolabelled glucose (using, for example, the Perkin Elmer 1450Microbeta JET reader). These assays are conducted as follows.

Materials and Methods: Prees Media

Complete media, also referred to as “Prees” media, is prepared asfollows. Dulbecco's Modified Eagle's Medium (DMEM) is supplemented withL-glutamine, penicillin-G and streptomycin (pen/strep), andheat-inactivated fetal bovine serum (FBS) (heat inactivated at65.degree. C. for 30 minutes). Because serum can affect the growth,adherence, and differentiation of cells, any new lot of serum was firsttested prior to use. Media was equilibrated in the incubator (5%CO.sub.2) until the pH was within the proper range (.about.7), asindicated by the red/orange color of the indicator dye. If the mediabecame pink (indicating a high pH), we discarded the media as basicconditions can affect cells and denature the insulin used in thedifferentiation medium-1 (DM1) and the differentiation medium-2 (DM2).

Differentiation Medias

Differentiation media-1 (DM1) was prepared by supplementing DMEM with10% FBS, L-glutamine, pen/strep, IBMX (375.mu.M), insulin (120 nM), anddexamethasone (188 nM). Differentiation media-2 (DM2) was prepared bysupplementing DMEM with 10% FBS, L-glutamine, pen/strep, and insulin(120 nM).

Preparation of Gelatinized Plates

Cell culture plates are gelatinized as follows. Gelatin (1% w/v indistilled water) was autoclaved and stored at room temperature. Thebottom of each cell culture well was covered uniformly in the gelatinsolution, ensuring that no bubbles are formed. This solution was removedleaving behind a thin film of gelatin. These plates are left to dryunder the tissue culture hood. Plates are next washed with PBS, afterwhich a 0.5% glutaric dialdehyde solution (glutaraldehyde in distilledwater) was added to the cell culture wells. After ten minutes, wells arewashed twice with DMEM containing pen-strep. Each washing step shouldlast for approximately five minutes.

Cell Culture

3T3-L1 pre-adipocyte cells are split approximately every 2-3 days orupon reaching a confluence of approximately 60%. Overconfluency mayaffect the ability of these cells to differentiate into adipocytes.

Other Reagents

D-(+)-glucose (“cold” glucose, not radiolabeled) was added to DPBS mixto a final concentration of 10 mM.

Lysis buffer, a mixture of a base (e.g., sodium hydroxide at a finalconcentration of 0.5N) and a detergent (e.g., sodium dodecyl sulphate(SDS) diluted to a final concentration of 0.1% w/v) was freshly preparedeach time (within one to two hours of use). Prior to use, lysis bufferwas warmed up to a temperature exceeding that of room temperature for aperiod of approximately 30 minutes to avoid precipitation of the buffer.

Determination of Glucose Uptake

Pre-adipocyte 3T3-L1 cells are plated at a density of approximately 5000cells/well (in black NUNC 96 well plate). These cells are differentiatedinto adipocytes in two separate steps. Initially, cells are cultured indifferentiation medium-1 (DM1) (day 1 of adipocyte differentation) for aperiod of two to three days. DM1 prevents proliferation and induces theexpression of adipocyte-specific genes. Cells are next cultured indifferentiation medium-2 (DM2) for 3 to 4 days, after which the culturemedia is replaced by fresh DM2. The glucose uptake assay is performed atday 9-15 of differentiation.

Two days prior to the experiment (at day 7-13 of differentiation), DM2is removed and replaced with fresh Prees media. Candidate compounds areadded at this time, allowing an incubation period of approximately 48hours. On the day of the experiment, cells (now at day 9 to 15 ofdifferentiation) are serum starved for three hours in DPBS, magnesiumsulfate (0.8 mM), and Hepes (10 mM) at pH .about.7. After thisincubation period, fresh DPBS containing insulin (10 nM) is added to theadipocytes. Fresh DPBS without any insulin are placed on cells thatserved as a negative control. Following an incubation period of 25minutes at 37.degree. C., radioactive glucose (labeled with .sup.14C, ata final concentration of 0.04 mM, .about.0.26.mu.Ci.sup.14C-glucose ineach well) is added to the media for a period of 15 minutes at roomtemperature. Media is next removed and cells are washed thoroughly andlysed. Upon lysis, cells form a small, cloudy mass, detached from thewell bottom. 10% glacial acetic acid is added to each well to neutralizethe lysis reaction. Scintillation fluid is next added to the wells andthe incorporation of glucose is determined by measuring the amount ofradioactivity in each well using the MicroBeta plate reader.

Using the foregoing experimental protocol, an Epi-shifter is identifiedas capable of treating a metabolic disorder, e.g., diabetes, when theEpi-shifter enhances, increases or augments insulin-stimulated glucoseuptake in the cells in vitro.

Example 16 Identification of a MIM Associated with an Metabolic Disorder

In order to evaluate a candidate molecule (e.g., environmentalinfluencer) as a potential MIM, the selected candidate MIM isexogenously added to a panel of cell lines, including both diseased(cancer) cell lines and normal control cell lines, and the changesinduced to the cellular microenvironment profile for each cell line inthe panel are assessed. Changes to cell morphology, physiology, and/orto cell composition, including for example, mRNA and protein levels, areevaluated and compared for the diseased cells as compared to normalcells.

Changes to cell morphology/physiology are evaluated by examining thesensitivity and apoptotic response of cells to the candidate MIM. Theseexperiments are carried out as described in detail in Example 3.Briefly, a panel of cell lines consisting of at least one control cellline and at least one cancer cell line are treated with variousconcentrations of the candidate MIM. The sensitivity of the cell linesto the potential MIM are evaluated by monitoring cell survival atvarious times, and over the range of applied concentrations. Theapoptoic response of the cell lines to the potential MIM are evaluatedby using, for example, Nexin reagent in combination with flow cytometrymethodologies. Nexin reagent contains a combination of two dyes, 7AADand Annexin-V-PE, and allows quantification of the population of cellsin early and late apoptosis. An additional apoptosis assay that measuressingle-stranded DNA may be used, using for example Apostrand™ ELISAmethodologies. The sensitivity and apoptotic response of the disease andcontrol cell lines are evaluated and compared. A molecule that displaysdifferential cytotoxicity and/or that differentially induces theapoptotic response in the diseased cells as compared to the normal cellsis identified as a MIM.

Changes in the composition of cells following treatment with thecandidate MIM are evaluated. Changes in gene expression at the mRNAlevel are analyzed using Real-Time PCR array methodology. Theseexperiments are carried out as described in detail in Examples 6 and9-13. Briefly, the candidate MIM is exogenously added to one or morecell lines including, for example a diseased cell and a normal controlcell line, and mRNA is extracted from the cells at various timesfollowing treatment. The level of mRNAs for genes involved in specificpathways are evaluated by using targeted pathway arrays, including, forexample, arrays specific for apoptosis, oxidative stress and antioxidatedefense, angiogenesis, heat shock or diabetes. The genes that arealtered in their mRNA transcription by a two-fold level or greater areidentified and evaluated. A molecule that induces changes in mRNA levelsin cells and/or that induces differential changes in the level of one ormore mRNAs in the diseased cells as compared to the normal cells isidentified as a MIM.

In complementary experiments, changes in gene expression at the proteinlevel are analyzed by using antibody microarray methodology,2-dimensional gel electrophoresis followed by protein identificationusing mass spectrometry characterization, and by western blot analysis.These experiments are carried out as described in detail in Examples 7,4 and 8, respectively. Briefly, the candidate MIM is exogenously addedto one or more cell lines, including, for example a diseased cell and anormal control cell line, and soluble protein is extracted from thecells at various times, e.g., 6 hours or 24 hours, following treatment.Changes induced to protein levels by the candidate MIM are evaluated byusing an antibody microarray containing antibodies for over 700proteins, sampling a broad range of protein types and potential pathwaymarkers. Further complementary proteomic analysis can be carried byemploying 2-dimensional (2-D) gel electrophoresis coupled with massspectrometry methodologies. The candidate MIM is exogenously added toone or more cell lines, including, for example a diseased cell and anormal control cell line, and cell pellets are lysed and subjected to2-D gel electrophoresis. The gels are analyzed to identify changes inprotein levels in treated samples relative to control, untreatedsamples. The gels are analyzed for the identification of spot changesover the time course of treatment due to increased levels, decreasedlevels or post-translational modification. Spots exhibitingstatistically significant changes are excised and submitted for proteinidentification by trypsin digestiona do mass spectrometrycharacterization. The characterized peptides are searched againstprotein databases with, for example, Mascot and MSRAT software analysisto identify the proteins. In addition to the foregoing 2-D gel analysisand antibody microarray experiments, potential changes to levels ofspecific proteins induced by the candidate MIM may be evaluated byWestern blot analysis. In all of the proteomic experiments, proteinswith increased or decreased levels in the various cell lines areidentified and evaluated. A molecule that induces changes in proteinlevels in cells and/or that induces differential changes in the level ofone or more proteins in the diseased cells as compared to the normalcells is identified as a MIM.

Genes found to be modulated by treatment with a candidate MIM from theforegoing experiments are subjected to cellular and biochemical pathwayanalysis and can thereby be categorized into various cellular pathways,including, for example apoptosis, cancer biology and cell growth,glycolysis and metabolism, molecular transport, and cellular signaling.

Experiments are carried out to confirm the entry of a candidate MIM intocells, to determine if the candidate MIM becomes localized within thecell, and to determine the level and form of the candidate MIM presentin the cells. These experiments are carried out, for example, asdescribed in detail in Example 5. For example, to determine the leveland the form of the candidate MIM present in the mitochondria,mitochondrial enriched preparations from cells treated with thecandidate MIM are prepared and analyzed. The level of the candidate MIMpresent in the mitochondria can thereby be confirmed to increase in atime and dose dependent manner with the addition of exogenous candidateMIM. In addition, changes in levels of proteins from mitochondriaenriched samples are analyzed by using 2-D gel electrophoresis andprotein identification by mass spectrometry characterization, asdescribed above for total cell protein samples. Candidate MIMs that arefound to enter the cell and to be present at increased levels, e.g., inthe mitochondria, are identified as a MIM. The levels of the candidateMIM in the cell, or, for example, specifically in the mitochondria, overthe time course examined can be correlated with other observed cellularchanges, as evidenced by, for example, the modulation of mRNA andprotein levels for specific proteins.

Candidate MIMs observed to induce changes in cell composition, e.g., toinduce changes in gene expression at the mRNA or protein level, areidentified as a MIM. Candidate MIMs observed to induce differentialchanges in cell morphology, physiology or cell composition (e.g.,differential changes in gene expression at the mRNA or protein level),in a disease state (e.g., diabetes or obestity) as compared to a normalstate are identified as a MIM and, in particular, as havingmultidimensional character. Candidate MIMs found to be capable ofentering a cell are identified as a MIM and, in particular, as havingmultidimensional character since the candidate MIM thereby exhibits acarrier effect in addition to a therapeutic effect.

Example 17 Identification of CoQ10 as an Epi-Shifter Associated with aMetabolic Disorder

A panel of skin cell lines consisting of a control cell lines (primaryculture of keratinocytes and melanocytes) and several skin cancers celllines (SK-MEL-28, a non-metastatic skin melanoma; SK-MEL-2, a metastaticskin melanoma; or SCC, a squamous cell carcinoma; PaCa2, a pancreaticcancer cell line; or HEP-G2, a liver cancer cell line) were treated withvarious levels of Coenzyme Q10. The cancer cell lines exhibited analtered dose dependent response when compared to the control cell lines,with an induction of apoptosis and cell death in the cancer cells only.Detailed exemplary experiments are presented in, e.g., Example 3 herein.

Assays were employed to assess changes in the mRNA and protein levelscomposition of the above-identified cells following treatment withCoQ10. Changes in mRNA expression were analyzed using real-time PCRmicroarrays specific for each of apoptosis, oxidative stress andantioxidants, angiogenesis and diabetes. Changes in protein expressionwere analyzed using antibody microarray analysis and western blotanalysis. The results from these assays demonstrated that significantchanges in gene expression, both at the mRNA and protein levels, wereoccurring in the cell lines due to the addition of the Coenzyme Q10.Numerous genes known to be associated with or involved in cellularmetabolic processes were observed to be modulated as a result oftreatment with CoQ10. For example, expression of the nuclear receptorprotein HNF4A was found to be upmodulated in cells following Q10treatment. Expression of transaldolase 1 (TAL) was also modulated incells treated with Q10. TAL balances the levels of NADPH and reactiveoxygen intermediate, thereby regulating the mitochondrialtrans-membrande potentional, which is a critical checkpoint of ATPsynthesis and cell survival. Of particular relevance to metabolicdisorders, numerous genes known to be associated with, e.g., diabetes,were identified as being regulated by Q10. Detailed exemplaryexperiments are presented in, e.g., Examples 4, 6, 7, 8 and 9 herein.

Q10 is an essential cofactor for exidative phosphorylation processes inthe mitochondria for energy production. The level of Coenzyme Q10, aswell as the form of CoQ10, present in the mitochondria was determined byanalyzing mitochondrial enriched preparations from cells treated withCoQ10. The level of Coenzyme Q10 present in the mitochondria wasconfirmed to increase in a time and dose dependent manner with theaddition of exogenous Q10. The time course correlated with a widevariety of cellular changes as observed in modulation of mRNA andprotein levels for specific proteins related to metabolic and apoptoticpathways. Detailed exemplary experiments are presented in, e.g., Example5 herein.

The results described herein identified the endogenous molecule CoQ10 asan epi-shifter. In particular, the results identified CoQ10 as inducinga shift in the metabolic state, and partially restoration ofmitochondrial function, in cells. These conclusions are based on thefollowing interpretation of the data described herein and the currentknowledge in the relevant art.

Q10 is known to be synthesized, actively transported to, enriched in,and utilized in the mitochondrial inner membrane. Q10 is also known tobe an essential cofactor for oxidative phosphorylation processes in themitochondrial for energy production. However, most cancer cellspredominantly produce energy by glycolysis followed by lactic acidfermentation in the cytosol, rather than by oxidation of pyruvate inmitochondria like most normal cells. The oxidative phosphorylationinvolves the electron transport complexes and cytochrome c. Apoptosisinvolves the disruption of the mitochondria, with permiabilization ofthe inter mitochondrial membrane by pro-apoptitic factors. By utilizinga different metabolic energy synthesis pathway, cancer cells are able tomitigate the normal apoptosis response to abnormalities in the cell.While not wishing to be bound by theory, Applicants propose that Q10 isfunctioning by upregulating the oxidative phosphorylation pathwayproteins, thus switching the mitochondrial function back to a state thatwould recognize the oncogenic defects and trigger apoptosis. Thus, Q10is acting as an Epi-shifter by shifting the metabolic state of a cell.

Example 18 Identification of an Epi-Shifter Associated with a MetabolicDisorder

A panel of skin cell lines consisting of control cell lines (e.g.,primary culture of keratinocytes and melanocytes) and cancer cell lines(e.g., SK-MEL-28, a non-metastatic skin melanoma; SK-MEL-2, a metastaticskin melanoma; or SCC, a squamous cell carcinoma; PaCa2, a pancreaticcancer cell line; or HEP-G2, a liver cancer cell line) are treated withvarious levels of a candidate Epi-shifter. Changes to cellmorphology/physiology are evaluated by examining the sensitivy andapoptotic response of cells to the candidate Epi-shifter. Theseexperiments are carried out as described in detail in Example 3.Briefly, the sensitivity of the cell lines to the candidate Epi-shifterare evaluated by monitoring cell survival at various times, and over arange of applied concentrations. The apoptoic response of the cell linesto the candidate Epi-shifter are evaluated by using, for example, Nexinreagent in combination with flow cytometry methodologies. Nexin reagentcontains a combination of two dyes, 7AAD and Annexin-V-PE, and allowsquantification of the population of cells in early and late apoptosis.An additional apoptosis assay that measures single-stranded DNA may beused, using for example Apostrand™ ELISA methodologies. The sensitivityand apoptotic response of the disease and control cell lines areevaluated and compared. Candidate Epi-shifters are evaluated based ontheir ability to inhibit cell growth preferentially or selectively incancer cells as compared to normal or control cells. CandidateEpi-shifters are further evaluated based on their ability topreferentially or selectively induce apoptosis in cancer cells ascompared to normal or control cells.

Assays are employed to assess changes in the mRNA and protein levelcomposition of the above-identified cells following treatment with thecandidate Epi-shifter. Changes in mRNA levels are analyzed usingreal-time PCR microarrays. These experiments are carried out asdescribed in detail in Examples 6 and 9-13. Briefly, mRNA is extractedfrom the cells at various times following treatment. The level of mRNAsfor genes involved in specific pathways are evaluated by using targetedpathway arrays, including, arrays specific for apoptosis, oxidativestress and antioxidate defense, angiogenesis, heat shock or diabetes.The genes that are altered in their mRNA transcription by a two-foldlevel or greater are identified and evaluated.

Changes in protein expression are analyzed using antibody microarrayanalysis, 2-D gel electrophoresis analysis coupled with massspectrometry characterization, and western blot analysis. Theseexperiments are carried out as described in detail in Examples 7, 4 and8, respectively. Briefly, soluble protein is extracted from the cells atvarious times, e.g., 6 hours or 24 hours, following treatment with thecandidate Epi-shifter. Changes induced to protein levels by thecandidate Epi-shifter are evaluated by using an antibody microarraycontaining antibodies for over 700 proteins, sampling a broad range ofprotein types and potential pathway markers. Further complementaryproteomic analysis can be carried out by employing 2-dimensional (2-D)gel electrophoresis coupled with mass spectrometry methodologies. Thecandidate Epi-shifter is exogenously added to the cell lines and cellpellets are lysed and subjected to 2-D gel electrophoresis. The gels areanalyzed to identify changes in protein levels in treated samplesrelative to control, untreated samples. The gels are analyzed for theidentification of spot changes over the time course of treatment due toincreased levels, decreased levels or post-translational modification.Spots exhibiting statistically significant changes are excised andsubmitted for protein identification by trypsin digestion and massspectrometry characterization. The characterized peptides are searchedagainst protein databases with, for example, Mascot and MSRAT softwareanalysis to identify the proteins. In addition to the foregoing 2-D gelanalysis and antibody microarray experiments, potential changes tolevels of specific proteins induced by the candidate MIM may beevaluated by Western blot analysis. In all of the proteomic experiments,proteins with increased or decreased levels in the various cell linesare identified and evaluated.

Candidate Epi-shifters are evaluated based on changes induced to geneexpression, at the mRNA and/or protein levels, in the cell lines due tothe addition of the candidate Epi-shifter. In particular, candidateEpi-shifters are evaluated based on their ability to modualate genesknown to be associated with or involved in cellular metabolic processes.Of particular relevance to metabolic disorders, candidate Epi-shiftersare evaluated based on their ability to modulate genes known to beassociated with, for example, diabetes or obesity.

The level of the candidate Epi-shifter, as well as the form of thecandidate Epi-shifter, present in the cell or a particular cell locationis determined using routine methods known to the skilled artisan. Forexample, the level of the candidate Epi-shifter in mitochondria overtime and over a range of doses is determined by analyzing mitochondrialenriched preparations from cells treated with the candidate Epi-shifter.The levels of the candidate Epi-shifter in the mitochondria over thetime course can be compared and correlated with other cellular changesobserved, such as modulation of mRNA and protein levels for specificproteins related to metabolic and apoptotic pathways.

Candidate Epi-shifters observed to induce a shift in the metabolic stateof a cell based on the results obtained from the foregoing experimentsare identified as Epi-shifters. For example, a candidate Epi-shifterthat enhances, increases or augments insulin-stimulated glucose uptakein cells is identified as an Epi-shifter.

Example 19 Identification of Vitamin D3 as an Epi-Shifter

Vitamin D3, or 1α, 25-dihydroxyvitamin D3 (also known as calcitriol), isa vitamin D metabolite that is synthesized from vitamin D by a two-stepenzymatic process. Vitamin D3 interacts with its ubiquitous nuclearvitamin D receptor (VDR) to regulate the transcription of a widespectrum of genes involved in calcium and phosphate homeostasis as wellas in cell division and differentiation. Vitamin D3 has been reported tohave anticancer effects in numerous model systems, including squamouscell carcinoma, prostate adenocarcinoma, cancers of the ovary, breastand lung (reviewed in Deeb et al. 2007 Nature Reviews Cancer 7:684-700).

The anticancer effects of vitamin D3 are reported to involve multiplemechanisms, including growth arrest at the G1 phase of the cell cycle,apoptosis, tumor cell differentiation, disruption of growthfactor-mediated cell survival signals, and inhibition of angiogenesisand cell adhesion (reviewed in Deeb et al. 2007 Nature Reviews Cancer7:684-700). For example, with particular respect to apoptosis, VitaminD3 has been reported to induce apoptosis by regulating key mediators ofapoptosis, such as repressing the expression of the anti-apoptotic,pro-survival proteins BCL2 and BCL-XL, or inducing the expression ofpro-apoptotic proteins (e.g., BAX, BAK and BAD) (Deeb et al. 2007). In afurther example, with particular respect to angiogenesis, Vitamin D3 hasbeen reported to inhibit the proliferation of some tumor-derivedendothelial cells and to inhibit the expression of vascular endothelialgrowth factor (VEGF) that induces angiogenesis in tumors (reviewed inMasuda and Jones, 2006 Mol. Cancer. Ther. 5(4): 797-8070). In anotherexample, with particular respect to cell cycle arrest, Vitamin D3 hasbeen reported to induce gene transcription of the cyclin-dependentkinase inhibitor p21WAFI/CIPI and to induce the synthesis and/orstabilization of the cyclin-dependent kinase inhibiotor p27KIPI protein,both of which are critical for induction of G1 arrest. (Deeb et al.2007).

Based on the foregoing observations, Vitamin D3 is identified as anEpi-shifter, i.e., owing to its ability to shift the metabolic state ofa cell. Vitamin D3 is an Epi-shifter owing to its ability to induceapoptosis in a cell and, in particular, based on its ability todifferentially inhibit cell growth and induce the apoptotic response indiseased (cancer) cells as compared to normal cells (e.g.,differentially modulate expression of proteins, such as BCL-2, BCL-XL,and BAX, involved in apoptosis in cancer cells as compared to normalcells).

Example 20 Western Analysis of Cells Treated with Coenzyme Q10

Over the past five decades enormous volume of information has beengenerated implicating endogenous/exogenous factors influencing specificprocesses as the underlying cause of malignant transformations. Clinicaland basic literature provides evidence that changes in the DNA structureand function play a significant role in the initiation and progressionof cancer, defining cancer as a genetic disease (Wooster, 2010; Haiman,2010). In the early 1920s, Otto Warburg and other investigators involvedin characterizing fundamental changes in etiology of oncogenesisdescribed two major observations (a) the ability of cells to transportand utilize glucose in the generation of ATP for energy production inthe presence of oxygen—also known as Warburg Effect and (b) alterationsin the mitochondrial structure and function—including changes in theelectron transport leading to a decrease in the production ofmitochondrial ATP. The past few years has seen a resurgence in theinvestigating the central role of cellular bioenergetics in the etiologyof cancer i.e. viewing cancer as a metabolic disease.

Historically, although mutations in genes has been thought to beresponsible for changes in gene expression, there is accumulatingliterature in support of epigenetic processes playing a critical role ininfluencing gene expression in supporting carcinogenesis. This isevidenced by the observation that mutation rate for most genes is lowand cannot account for the numerous (spectrum of) mutations found in thecancer cells. Epigenetic alteration is regulated by methylation andmodification of histone tails, both changes inherently linked to theenergy (nutrient) status of the cells since they require theavailability of co-factors e.g. acetyl CoA requirement for histoneacetylation (ref). The biosynthesis of acetyl CoA depends on glycolysisand Kreb's Cycle, directly linking the intracellular energy status toregulation of gene expression and activity.

In normal cells, mitochondrial oxidative phosphorylation generatessufficient

ATP to meet the energy demands for maintaining normal physiologicalactivities and cell survival. A consequence of mitochondrial energyproduction is the generation of reactive oxygen species (ROS), aberrantproduction of which leads to damage of mitochondria (refs). It is wellestablished that chronic ROS generation by the mitochondria leads tocumulative accumulation of genetic mutations, a phenomenon that has beenimplicated in the etiology of carcinogenesis. It has been suggested thatcancer cells decrease mitochondrial respiration to minimize ROSgeneration, and switch to glycolysis to sustain energy production. Thus,a progressive shift of energy generation from oxidative phosphorylationto glycolysis would be essential for a cell to maintain energyproduction to maintain physiological functions and could be associatedwith the progression of a normal cell phenotype to that of a cancercell. The progressive shift in cellular energy (bioenergetic) profile intandem with accumulated alteration (mutations) in mitochondrial geneticmake-up alters the cellular metabolome. Changes in the whole cellmetabolomic profile as a consequence of mitochondrial phosphorylation toglycolysis transition corresponds to an abnormal bioenergetic inducedmetabolomic profile and is the underlying cause supportingcarcinogenesis. Targeted intervention using an endogenous molecule toelicit a cellular metabolomic shift towards conditions of anon-cancerous normal mitochondrial oxidative phosphorylation associatedcellular bioenergetic state represents a therapeutic endpoint in thetreatment of cancer.

Coenzyme Q10 as a MIM Causing an Epi-Metabolomic Shift

The data presented herein demonstrates that treatment of normal andcancer cells with Coenzyme Q10 is associated with changes in theexpression of proteins that regulate key biochemical terminals withinthe glycolysis—mitochondrial oxidative stress continuum. The combinationof data describing assessment of protein expression by western blottingand oxygen consumption rates demonstrates that in normal cells, there isno significant alteration in normal glycolytic and mitochondrialrespiration rates following exposure to Coenzyme Q10. Thus, the valuesfor expression of the proteins and mitochondrial respiration rates innormal cell lines e.g. HDFa (normal human adult fibroblast), HASMC(normal human aortic smooth muscle cell), nFib (normal fibroblast) andHeKa (normal human keratinocytes) can be considered as representativesof baseline physiological state. Any deviation in expression of proteinsand mitochondrial respiration rates in cancer cell lines, e.g. HepG2(liver cancer), PaCa-2 (pancreatic cancer), MCF7 (breast cancer), SK-MEL(melanoma) and SCC-25 (squamous cell carcinoma), is representative ofalteration due to initiation/progression of the disease, in this casecancer. The experimental evidence provides support to the hypothesisthat exposure of Coenzyme Q10 to cancer cells is associated withcellular pathophysiological reorganization that is reminiscent of normalcells. Specifically, the data provided herein demonstrates that CoenzymeQ10 exposure in cancer cells is associated with a shift in theglycolytic pathways and mitochondrial oxidative phosphorylationresponsible for induction of global reorganization of cellulararchitecture to that observed in normal cells.

In normal cells, the end-points of glycolytic output are linked tomitochondrial oxidative phosphorylation (OXPHOS), i.e. generation ofpyruvate from glucose via the glycolytic pathway for the entry into theKreb's Cycle (also known as Tricarboxylic acid cycle, TCA, or CitricAcid Cycle) to generate reducing equivalents to support themitochondrial OXPHOS for ATP production. Thus, in normal cells theexpression and functional orientation of gene products involved inglycolysis is primed towards adequate generation of pyruvate and itsentry into the Kreb's Cycle. Dysregulated expression and function of keyproteins participating in glycolysis and Kreb's Cycle pathways in cancercells results in enhanced glycolysis with a significant decrease inmitochondrial function. Exposure of cancer cells to Coenzyme Q10, anendogenous molecule that selectively influences the mitochondrialrespiratory chain, alters (normalizes) expression of proteins of theglycolyis and Kreb's Cycle pathways to facilitate a bioenergetic shiftsuch that energy production (i.e. ATP generation) is restored to themitochondria.

EXPERIMENTAL PROCEDURE Western Blot Experiment 1

The cells that were used for the experiment were HDFa, and MCF-7 cellsthat were treated or not with Coenzyme Q10 at two differentconcentrations, 50 μM and 100 μM, and harvested after 24 hours oftreatment. The whole cell pellets were resuspended one at a time in 1 mLof C7 buffer and transferred to labeled 15 mL tubes. The samples werethen sonicated in the cold room on ice using 6 sonic pulses with thesetting at #14. The samples were spun for a short time to 2500 g aftersonication and the samples transferred to 2 ml tubes. The pH wasverified of each sample (pH should be 9.0) using the foam remaining inthe 50 mL sample tubes.

Alkylation and reduction of samples was performed for each sample byadding 10 ul of 1M acrylamide, 25 ul of tributylphoshene and incubationfor 90 mins with intermittent mixing. After incubation, 10 ul of 1M DTTwas added and the tubes were spun at 20,000 g at 20 deg C. for 10minutes and transferred the supernatant to labeled Amicon Ultracentrifugal filter units with a 10 k cut off (Millipore catalog #UFC801024). The samples were spun for 15 minutes at 2500 g in 2 intervals.The conductivity was measured for Chaps alone as well as the samplesusing a conductivity meter. If the conductivity of samples is high, then1 ml of chaps was added for buffer exchange and spun again at 2500 guntil the volume was down to 250 ul. When the conductivity was 200 orless the samples were spun in 5 min intervals at 2500 g until the volumeof the supernatant was between 150-100 ul. The sample supernatants weretransferred to eppendorf tubes and Bradford assay was performed usingBSA as standard.

The samples were processed as per standard protocols as described aboveand the amount of protein in each of the samples was determined byBradford assay. Sample volumes equivalent to 10 ug of protein wereprepared as shown below with Lamelli Loading dye (LDS) and MilliQ waterwere run on a 4-12% Bis-Tris Novex NuPAGE gel (Invitrogen, cat#NP0323Box)

The gels were run for 50 minutes using 1× MOPS buffer using a NOVEXXcell Surelock system at 200 V. The gels were then transferred for 1hour using a NOVEX Xcell Surelock wet transfer protocol at 30 V. Theblots were stained with Simply Blue Safestain from Invitrogen (LC6065).

IDH1 and ATP Citrate Lyase Levels in HDFa and MCF-7 Samples.

After transfer each of the blots was placed in between 2 Whatman Filterpapers and dried for 15-20 minutes. After drying the blots were labeledwith the date, the type of samples and either blot 1 or blot 2 using aHB pencil. The molecular weight markers were outlined with the penciland with single lines for the blue and a doublet for the coloredmarkers. The blots were activated with methanol for 5 seconds, washedwith water for 5 minutes, and TBST for 15 minutes. The blots wereblocked for 1 hour with 5% blocking reagent in TBS-T at room temperatureand then washed 3 times with TBS-T (1×-15′; 2×5′ each). Blot 1 wasprobed with the primary antibody for IDH1 (Cell Signaling #3997) in TBSTwith 5% BSA (at 1:1000 dilutions) and blot 2 with the rabbit polyclonalantibody for ATP Citrate Lyase in 5% BSA (Cell Signaling #4332) at1:1000 dilution by incubation overnight at 4 deg C. with shaking. Afterthe overnight incubation with primary antibodies, the blots were washed3 times with TBS-T (1×-15′; 2×5′ each) and probed with the secondaryantibody (antirabbit; 1:10,000 dilution) for 1 h on the orbital tiltingshaker at room temperature. After 1 h of incubation with secondaryantibodies, the blots were washed 3 times with TBS-T (1×-15′; 2×5′ each)and then incubated with ECF reagent for 5 mins and then each blotscanned with 5100 Fuji Laser scanner at 25 uM resolution, 16 bit, greenlaser, at 400V and at 500 V.

Actin Levels in HDFa and MCF-7 Samples.

The above blots were stripped by incubating for 30 minutes withmethanol, followed by two 10 minute washes with TBS-T, then 30 minutesof incubation with Stripping buffer at 50 deg C., and followed by twowashes with 100 ml or more of TBS-T for 30′ each. The 2 blots werescanned in laser scanner to check for complete stripping. The blots werethen activated with methanol for 5 seconds, washed with water for 5minutes, and TBST for 15 minutes. The blots were blocked for 1 hour with5% blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1×-15′; 2×5′ each) and probed with the antibody for Actin in5% BSA (Sigma catalog #A5316, clone AC-74) at 1:5000 dilutions for 1hour at room temperature with shaking. After 1 hour of incubation withprimary antibody for Actin, the membranes were washed 3 times with TBS-T(1×-15′; 2×5′ each) and probed with the secondary antibody (antimouse;1:10,000 dilution) for 1 h on the orbital tilting shaker at roomtemperature. After 1 h of incubation with secondary antibodies, theblots were washed 3 times with TBS-T (1×-15′; 2×5′ each) and thenincubated with ECF reagent for 5 minutes and then each blot scanned with5100 Fuji Laser scanner at 25 uM resolution, 16 bit, green laser, at400V and at 500 V.

Western Blot Experiment 2

The cells used in this experiment were SKMEL28, SCC-25, nFib and Hekathat were treated or not with coenzyme Q10 at two differentconcentrations, 50 μM or 100 μM, and harvested after 3, 6 and/or 24hours of treatment. The samples were processed and run on a 4-12%Bis-Tris Novex NuPAGE gel as described above. The gels were run,transferred and stained essentially as described above.

Levels of IDH1 for the 4 Cell Lines

After transfer the blot was dried for 15-20 minutes, activated withmethanol for 5 seconds, washed with water for 5 minutes, and TBST for 15minutes. The blot was blocked for 1 hour with 5% blocking reagent inTBS-T at room temperature and then washed 3 times with TBS-T (1×-15′;2×5′ each). This was then probed with the primary antibody for IDH1(Cell Signaling #3997) in TBST with 5% BSA (at 1:1000 dilutions) byincubation overnight at 4 deg C. with shaking. After the overnightincubation with primary antibody for IDH1, the blot was washed 3 timeswith TBS-T (1×-15′; 2×5′ each) and probed with the secondary antibody(antirabbit; 1:10,000 dilution) for 1 h at room temperature. After 1 hof incubation with secondary antibodies, the blot was washed 3 timeswith TBS-T (1×-15′; 2×5′ each) and then incubated with ECF reagent for 5mins and then each blot scanned with 5100 Fuji Laser scanner at 25 uMresolution, 16 bit, green laser, at 400V and at 500 V.

ATP Citrate Lyase Levels in 4 Different Cell Lines.

The Isocitrate dehydrogenase blot was stripped by incubating for 30minutes with methanol, followed by two 10 minute washes with TBS-T, then30 minutes of incubation with stripping buffer at 50 deg C., andfollowed by two washes with 100 ml or more of TBS-T for 30′ each. Theblot was scanned in laser scanner to check for complete stripping. Theblot was activated with methanol for 5 seconds, washed with water for 5minutes, and TBST for 15 minutes. The blot was blocked for 1 hour with5% blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1×-15′; 2×5′ each). This was then probed with the rabbitpolyclonal antibody for ATP Citrate Lyase in 5% BSA (Cell Signaling#4332) at 1:1000 dilution overnight at 4 deg C. with shaking. After theovernight incubation with primary antibody for ATP Citrate Lyase, themembrane was washed 3 times with TBS-T (1×-15′; 2×5′ each) and probedwith the secondary antibody (antirabbit; 1:10,000 dilution) for 1 h onthe orbital tilting shaker at room temperature. After 1 h of incubationwith secondary antibodies, the blot was washed 3 times with TBS-T(1×-15′; 2×5′ each) and then incubated with ECF reagent for 5 minutesand then each blot scanned with 5100 Fuji Laser scanner at 25 uMresolution, 16 bit, green laser, at 400V and at 500 V.

Actin Levels in 4 Different Cell Lines.

The ATP Citrate Lyase blot was stripped by incubating for 30 minuteswith methanol, followed by two 10 minute washes with TBS-T, then 30minutes of incubation with Stripping buffer at 50 deg C., and followedby two washes with 100 ml or more of TBS-T for 30′ each. The blot wasscanned in laser scanner to check for complete stripping. The blot wasactivated with methanol for 5 seconds, washed with water for 5 minutes,and TBST for 15 minutes. The blot was blocked for 1 hour with 5%blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1×-15′; 2×5′ each) and probed with the antibody for Actin in5% BSA (Sigma catalog #A5316, clone AC-74) at 1:5000 dilutions for 1hour at room temperature with shaking. After 1 hour of incubation withprimary antibody for Actin, the membranes were washed 3 times with TBS-T(1×-15′; 2×5′ each) and probed with the secondary antibody (antimouse;1:10,000 dilution) for 1 h on the orbital tilting shaker at roomtemperature. After 1 h of incubation with secondary antibodies, theblots were washed 3 times with TBS-T (1×-15′; 2×5′ each) and thenincubated with ECF reagent for 5 minutes and then each blot scanned with5100 Fuji Laser scanner at 25 uM resolution, 16 bit, green laser, at400V and at 500 V.

Western Blot Experiment 3

The cells used in this experiment were HepG2, HASMC, and PACA2 cellsthat were treated or not with Coenzyme Q10 at two differentconcentrations (50 μM and 100 μM) and harvested 48 hours of treatment.In this experiment (western blot experiment 3), and in all of theexperiments described below in this Example (i.e., western blotexperiments 4 through 9), the cells were additionally treated witheither 5 mM glucose (“5G”) or 22 mM glucose (“22G”). The samples derivedfrom the cells were processed and run on a 4-12% Bis-Tris Novex NuPAGEgel as described above. The gels were run, transferred and stainedessentially as described above.

IDH1, ATP Citrate Lyase and Actin Levels in HASMC vs. PACA2 and HepG2.

The levels of IDH1, ATP citrate lyase and actin levels were determinedby probing the blots with primary antibodies for IDH1, ATP citrate lyaseand actin, essentially as described above.

Western Blot Experiment 4

The cells used in this experiment were HepG2 cells that were treated ornot with Coenzyme Q10 at two different concentrations, 50 or 100 μM, andharvested after 24 or 48 hours of treatment. The samples were processedand run on a 4-12% Bis-Tris Novex NuPAGE gel as described above. Thegels were run, transferred and stained essentially as described above.

Lactate Dehydrogenase Levels in HepG2 Cells.

After transfer each blot was dried for 15-20 minutes, activated withmethanol for 5 seconds, washed with water for 5 minutes, and TBST for 15minutes. The blots were blocked for 1 hour with 5% blocking reagent inTBS-T at room temperature and then washed 3 times with TBS-T (1×-15′;2×5′ each) and probed with the primary antibody for LactateDehydrogenase (abcam ab2101; polyclonal) in 5% BSA (at 1:1000 dilutions)by incubation overnight at 4 deg C. with shaking. After the overnightincubation with primary antibody for Lactate Dehydrogenase, the blotswere washed 3 times with TBS-T (1×-15′; 2×5′ each) and probed with thesecondary antibody (rabbit antigoat; 1:10,000 dilution) for 1 h at roomtemperature. After 1 h of incubation with secondary antibodies, theblots were washed 3 times with TBS-T (1×-15′; 2×5′ each) and thenincubated with ECF reagent for 5 mins and then each blot scanned with5100 Fuji Laser scanner at 25 uM resolution, 16 bit, green laser, at400V and at 500 V.

Pyruvate Kinase Muscle form (PKM2) Levels in HepG2 Cells.

The lactate dehydrogenase blots were stripped by incubating for 30minutes with methanol, followed by two 10 minute washes with TBS-T, then30 minutes of incubation with Stripping buffer at 50 deg C., andfollowed by two washes with 100 ml or more of TBS-T for 30′ each. The 2blots were scanned in laser scanner to check for complete stripping. Theblots were activated with methanol for 5 seconds, washed with water for5 minutes, and TBST for 15 minutes. The blots were blocked for 1 hourwith 5% blocking reagent in TBS-T at room temperature and then washed 3times with TBS-T (1×-15′; 2×5′ each) and probed with the rabbitpolyclonal antibody for Pyruvate Kinase M2 in 5% BSA (NOVUS BIOLOGICALScatalog #H00005315-DO1P) at 1:500 dilution overnight at 4 deg C. withshaking. After the overnight incubation with primary antibody forPyruvate Kinase M2, the membranes were washed 3 times with TBS-T(1×-15′; 2×5′ each) and probed with the secondary antibody (antirabbit;1:10,000 dilution) for 1 h on the orbital tilting shaker at roomtemperature. After 1 h of incubation with secondary antibodies, theblots were washed 3 times with TBS-T (1×-15′; 2×5′ each) and thenincubated with ECF reagent for 5 minutes and then each blot scanned with5100 Fuji Laser scanner at 25 uM resolution, 16 bit, green laser, at400V and at 500 V.

Pyruvate Dehydrogenase Beta Levels in HepG2 Cells.

The pyruvate kinase blots were stripped by incubating for 30 minuteswith methanol, followed by two 10 minute washes with TBS-T, then 30minutes of incubation with Stripping buffer at 50 deg C., and followedby two washes with 100 ml or more of TBS-T for 30′ each. The 2 blotswere scanned in laser scanner to check for complete stripping. Aftermaking sure stripping of the antibody and the ECF reagent has worked,the blots were activated with methanol for 5 seconds, washed with waterfor 5 minutes, and TBST for 15 minutes. The blots are blocked for 1 hourwith 5% blocking reagent in TBS-T at room temperature and then washed 3times with TBS-T (1×-15′; 2×5′ each) and probed with the antibody forPyruvate Dehydrogenase in 5% BSA (ABNOVA catalog #H00005162-M03) at1:500 dilutions) overnight at 4 deg C. with shaking. After the overnightincubation with primary antibody for Pyruvate Dehydrogenase, themembranes were washed 3 times with TBS-T (1×-15′; 2×5′ each) and probedwith the secondary antibody (antimouse; 1:10,000 dilution) for 1 h onthe orbital tilting shaker at room temperature. After 1 h of incubationwith secondary antibodies, the blots were washed 3 times with TBS-T(1×-15′; 2×5′ each) and then incubated with ECF reagent for 5 minutesand then each blot scanned with 5100 Fuji Laser scanner at 25 uMresolution, 16 bit, green laser, at 400V and at 500 V.

Actin Levels in HepG2 Cells.

The Pyruvate Dehydrogenase blots were stripped and then reprobed foractin, essentially as described above.

Western Blot Experiment 5

The cells used in this experiment were MIAPACA2 (PACA2) cells that weretreated or not with Coenzyme Q10 at two different concentrations, 50 or100 μM, and harvested after 24 or 48 hours of treatment. The PACA2samples were processed and the gels were run, transferred, stained andscanned essentially as described above.

Lactate Dehydrogenase (LDH) and Pyruvate Dehydrogenase (PDH) Levels inPaCa2 Cells

The levels of LDH and PDH were determined by probing the blotssuccessively with primary antibodies for LDH and PDH, essentially asdescribed above.

Caspase 3 Levels in PaCa2 Cells.

The blots were stripped by incubating for 30 minutes with methanol,followed by two 10 minute washes with TBS-T, then 30 minutes ofincubation with Stripping buffer at 50 deg C., and followed by twowashes with 100 ml or more of TBS-T for 30′ each. The 2 blots werescanned in laser scanner to check for complete stripping. The blots wereactivated with methanol for 5 seconds, washed with water for 5 minutes,and TBST for 15 minutes. The blots were blocked for 1 hour with 5%blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1×-15′; 2×5′ each) and probed with the antibody for Caspase3 in 5% BSA (Santacruz Biotechnology # sc7272) at 1:200 dilutions)overnight at 4 deg C. with shaking. After the overnight incubation withprimary antibody for Caspase 3, the membranes were washed 3 times withTBS-T (1×-15′; 2×5′ each) and probed with the secondary antibody(antimouse; 1:10,000 dilution) for 1 h on the orbital tilting shaker atroom temperature. After 1 h of incubation with secondary antibodies, theblots were washed 3 times with TBS-T (1×-15′; 2×5′ each) and thenincubated with ECF reagent for 5 minutes and then each blot scanned with5100 Fuji Laser scanner at 25 uM resolution, 16 bit, green laser, at400V and at 500 V.

Western Blot Experiment 6

The cells that were used for this Western blot experiment were PC-3,HepG2, MCF-7, HDFa and PACA2 that were treated or not with a CoenzymeQ10 IV formulation and harvested after 24 hours of treatment. Thesamples were processed and the gels were run, transferred, stained andscanned essentially as described above.

Capase 3 and Actin Levels in Different Cell Types.

The levels of Caspase 3 and actin were determined by probing the blotssuccessively with primary antibodies for Caspase 3 and actin,essentially as described above.

Western Blot Experiment 7

The cells used in this experiment were Human Aortic Smooth Muscle(HASMC) cells that were treated or not with Coenzyme Q10 at twodifferent concentrations, 50 μM or 100 μM, and harvested after 24 or 48hours of treatment. The HASMC samples were processed and the gels wererun, transferred, stained and scanned essentially as described above.

Experimental Protocol for Actin:

The levels of actin were determined by probing the blots with a primaryantibody for actin, essentially as described above.

Experimental Protocol for Hif 1Alpha, Caspase3 and PDHB:

The Actin blots were stripped by incubating for 30 minutes withmethanol, followed by two 10 minute washes with TBS-T, then 30 minutesof incubation with Stripping buffer at 50 deg C., and followed by twowashes with 100 ml or more of TBS-T for 30′ each. The blots were scannedin laser scanner to check for complete stripping. The blots wereactivated with methanol for 5 seconds, washed with water for 5 minutes,and TBST for 15 minutes. The blots were blocked for 1 hour with 5%blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1×-15′; 2×5′ each) and probed with the primary antibody forHif 1 alpha, Caspase 3 or PDHB in 5% BSA (at 1:200 by incubationovernight at 4 deg C. with gentle shaking. The primary antibody for Hif1 alpha (Abcam ab2185; antirabbit) was at 1:500 dilution in 5% BSA. Theprimary antibody for Caspase 3 (Santacruz sc7272; antirabbit) was at1:200 dilution in 5% BSA. The primary antibody for PyruvateDehydrogenase beta (PDHB) (Novus Biologicals H00005162-M03; antimouse)was at 1:500 dilution in 5% BSA. After incubation with primaryantibodies, the membranes were washed 3 times with TBS-T (1×-15′; 2×5′each) and probed with the secondary antibody (PDHB antimouse; Hif 1a andCaspase 3 antirabbit; 1:10,000 dilution) for 1 h at room temperature.After 1 h of incubation with secondary antibodies, the blots were washed3 times with TBS-T (1×-15′; 2×5′ each) and then incubated with ECFreagent for 5 minutes and then each blot scanned with 5100 Fuji Laserscanner at 25 uM resolution, 16 bit, green laser, at 400V and at 500 V.

Experimental Protocol for PKM2, SDHB and SDHC:

The above blots were stripped by incubating for 30 minutes withmethanol, followed by two 10 minute washes with TBS-T, then 30 minutesof incubation with Stripping buffer at 50 deg C., and followed by twowashes with 100 ml or more of TBS-T for 30′ each. The blots were scannedin laser scanner to check for complete stripping. The blots wereactivated with methanol for 5 seconds, washed with water for 5 minutes,and TBST for 15 minutes. The blots were blocked for 1 hour with 5%blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1×-15′; 2×5′ each) and probed with the primary antibody forPKM2, SDHB or SDHC in 5% BSA in TBS-T by incubation overnight at 4 degC. with gentle shaking. The primary antibody for SDHC (ABNOVAH00006391-MO2; antimouse) was at 1:500 dilution. The primary antibodyfor SDHB was from Abcam ab4714-200; antimouse; at 1:1000 dilution. Theprimary antibody for Pyruvate Kinase M2 (PKM2) was from NovusBiologicals H00005315-D0IP; antirabbit; at 1:500 dilution. Afterincubation with primary antibodies, the membranes were washed 3 timeswith TBS-T (1×-15′; 2×5′ each) and probed with the secondary antibody(SDHB & C antimouse; and PKM2 antirabbit; 1:10,000 dilution) for 1 h onthe orbital tilting shaker at room temperature. After 1 h of incubation,the blots were washed 3 times with TBS-T (1×-15′; 2×5′ each) andincubated with ECF reagent for 5 minutes and then each blot scanned with5100 Fuji Laser scanner at 25 uM resolution, 16 bit, green laser, at400V and at 500 V.

Experimental Protocol for LDH and Bik:

The above blots were stripped by incubating for 30 minutes withmethanol, followed by two 10 minute washes with TBS-T, then 30 minutesof incubation with Stripping buffer at 50 deg C., and followed by twowashes with 100 ml or more of TBS-T for 30′ each. The blots were scannedin laser scanner to check for complete stripping. The blots wereactivated with methanol for 5 seconds, washed with water for 5 minutes,and TBST for 15 minutes. The blots were blocked for 1 hour with 5%blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1×-15′; 2×5′ each) and probed with the primary antibody forLDH or Bik in 5% BSA in TBS-T by incubation overnight at 4 deg C. withgentle shaking. The primary antibody for LDH was from Abcam ab2101;antigoat; at 1:1000 dilution. The primary antibody for Bik was from CellSignaling #9942; antirabbit; at 1:1000 dilution. After incubation withprimary antibodies, the membranes were washed 3 times with TBS-T(1×-15′; 2×5′ each) and probed with the secondary antibody (LDHantigoat; Jackson Laboratories) and Bik antirabbit; 1:10,000 dilution)for 1 h on the orbital tilting shaker at room temperature. After 1 h ofincubation, the blots were washed 3 times with TBS-T (1×-15′; 2×5′ each)and incubated with ECF reagent for 5 minutes and then each blot scannedwith 5100 Fuji Laser scanner at 25 uM resolution, 16 bit, green laser,at 400V and at 500 V.

Western Blot Experiment 9

The cells used were HepG2 cells that were treated or not with CoenzymeQ10 at two different concentrations, 50 μM or 100 μM, and harvestedafter 24 or 48 hours of treatment. The HepG2 samples processed and thegels were run, transferred, stained and scanned essentially as describedabove.

Experimental Protocol for Actin:

The levels of actin were determined by probing the blots with a primaryantibody for actin, essentially as described above.

Experimental Protocol for Caspase3 and MMP-6:

The Actin blots were stripped by incubating for 30 minutes withmethanol, followed by two 10 minute washes with TBS-T, then 30 minutesof incubation with Stripping buffer at 50 deg C., and followed by twowashes with 100 ml or more of TBS-T for 30′ each. The blots wereactivated with methanol for 5 seconds, washed with water for 5 minutes,and TBST for 15 minutes. The blots were blocked for 1 hour with 5%blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1×-15′; 2×5′ each) and probed with the primary antibody forCaspase 3 or MMP-6 in 5% BSA by incubation overnight at 4 deg C. withgentle shaking. The primary antibody for Caspase 3 (Abcam ab44976-100;antirabbit) was at 1:500 dilution in 5% BSA. The primary antibody forMMP-6 (Santacruz scMM0029-ZB5; antimouse) was at 1:100 dilution in 5%BSA. After incubation with primary antibodies, the membranes were washed3 times with TBS-T (1×-15′; 2×5′ each) and probed with the secondaryantibody (MMP-6 antimouse; Caspase 3 antirabbit; 1:10,000 dilution) for1 h at room temperature. After 1 h of incubation with secondaryantibodies, the blots were washed 3 times with TBS-T (1×-15′; 2×5′ each)and then incubated with ECF reagent for 5 minutes and then each blotscanned with 5100 Fuji Laser scanner at 25 uM resolution, 16 bit, greenlaser, at 400V and at 500 V.

Experimental Protocol for LDH:

The above blots were stripped by incubating for 30 minutes withmethanol, followed by two 10 minute washes with TBS-T, then 30 minutesof incubation with stripping buffer at 50 deg C., and followed by twowashes with 100 ml or more of TBS-T for 30′ each. The blots wereactivated with methanol for 5 seconds, washed with water for 5 minutes,and TBST for 15 minutes. The blots ere blocked for 1 hour with 5%blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1×-15′; 2×5′ each) and probed with the primary antibody forLDH in 5% BSA or 5% milk by incubation overnight at 4 deg C. with gentleshaking. The primary antibody for LDH 080309b1 (Abcam ab2101; antigoat)was at 1:1000 dilution in 5% BSA. The primary antibody for LDH 080309b2(Abcam ab2101; antigoat) was at 1:1000 dilution in 5% milk. Afterincubation with primary antibodies, the membranes were washed 3 timeswith TBS-T (1×-15′; 2×5′ each) and probed with the secondary antibody(Jackson Immuno Research antigoat; 1:10,000 dilution; 305-055-045) for 1h. After 1 h of incubation with secondary antibodies, the blots werewashed 3 times with TBS-T (1×-15′; 2×5′ each) and then incubated withECF reagent for 5 minutes and then each blot scanned with 5100 FujiLaser scanner at 25 uM resolution, 16 bit, green laser, at 400V and at500 V.

Experimental Protocol for Transaldolase and Hif1a:

The above blots were stripped by incubating for 30 minutes withmethanol, followed by two 10 minute washes with TBS-T, then 30 minutesof incubation with Stripping buffer at 50 deg C., and followed by twowashes with 100 ml or more of TBS-T for 30′ each. The blots wereactivated with methanol for 5 seconds, washed with water for 5 minutes,and TBST for 15 minutes. The blots are blocked for 1 hour with 5%blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1×-15′; 2×5′ each) and probed with the primary antibody forTransaldolase or Hifla in 5% BSA by incubation overnight at 4 deg C.with gentle shaking. The primary antibody for Transaldolase (Abcamab67467; antimouse) was at 1:500 dilution. The primary antibody forHifla (Abcam ab2185; antirabbit) was at 1:500 dilution. After incubationwith primary antibodies, the membranes were washed 3 times with TBS-T(1×-15′; 2×5′ each) and probed with the secondary antibody (antimouse orantirabbit; 1:10,000 dilution) for 1 h on the orbital tilting shaker atroom temperature. After 1 h of incubation with secondary antibodies, theblots were washed 3 times with TBS-T (1×-15′; 2×5′ each) and thenincubated with ECF reagent for 5 minutes and then each blot scanned with5100 Fuji Laser scanner at 25 uM resolution, 16 bit, green laser, at 400& 500V.

Experimental Protocol for IGFBP3 and TP53:

The above blots were stripped by incubating for 30 minutes withmethanol, followed by two 10 minute washes with TBS-T, then 30 minutesof incubation with Stripping buffer at 50 deg C., and followed by twowashes with 100 ml or more of TBS-T for 30′ each. The blots wereactivated with methanol for 5 seconds, washed with water for 5 minutes,and TBST for 15 minutes. The blots are blocked for 1 hour with 5%blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1×-15′; 2×5′ each) and probed with the primary antibody forIGFBP3 or TP53 in 5% BSA by incubation overnight at 4 deg C. with gentleshaking. The primary antibody for IGFBP3 (Abcam ab76001; antirabbit) wasat 1:100 dilution. The primary antibody for TP53 (Sigma Aldrich AV02055;antirabbit) was at 1:100 dilution. After incubation with primaryantibodies, the membranes were washed 3 times with TBS-T (1×-15′; 2×5′each) and probed with the secondary antibody (antirabbit; 1:10,000dilution) for 1 h on the orbital tilting shaker at room temperature.After 1 h of incubation with secondary antibodies, the blots were washed3 times with TBS-T (1×-15′; 2×5′ each) and then incubated with ECFreagent for 5 minutes and then each blot scanned with 5100 Fuji Laserscanner at 25 uM resolution, 16 bit, green laser, at 400 & 500V.

Experimental Protocol for Transaldolase and PDHB:

The above blots were stripped by incubating for 30 minutes withmethanol, followed by two 10 minute washes with TBS-T, then 30 minutesof incubation with Stripping buffer at 50 deg C., and followed by twowashes with 100 ml or more of TBS-T for 30′ each. The blots wereactivated with methanol for 5 seconds, washed with water for 5 minutes,and TBST for 15 minutes. The blots were blocked for 1 hour with 5%blocking reagent in TBS-T at room temperature and then washed 3 timeswith TBS-T (1×-15′; 2×5′ each) and probed with the primary antibody forTransaldolase or PDHB in 5% BSA by incubation overnight at 4 deg C. withgentle shaking. The primary antibody for Transaldolase (Santacruzsc51440; antigoat) was at 1:200 dilution. The primary antibody for PDHB(Novus Biologicals H00005162-M03; antimouse) was at 1:500 dilution.After incubation with primary antibodies, the membranes were washed 3times with TBS-T (1×-15′; 2×5′ each) and probed with the secondaryantibody (antigoat or antimouse; 1:10,000 dilution) for 1 h on theorbital tilting shaker at room temperature. After 1 h of incubation withsecondary antibodies, the blots were washed 3 times with TBS-T (1×-15′;2×5′ each) and then incubated with ECF reagent for 5 minutes and theneach blot scanned with 5100 Fuji Laser scanner at 25 uM resolution, 16bit, green laser, at 400 & 500V.

Results Isocitrate Dehydrogenase-1 (IDH-1)

Isocitrate dehydrogenase is one of the enzymes that is part of the TCAcycle that usually occurs within the mitochondrial matrix. However, IDH1is the cytosolic form of the enzyme that catalyzes the oxidativedecarboxylation of isocitrate to α-ketoglutarate and generates carbondioxide in a two step process. IDH1 is the NADP dependent form that ispresent in the cytosol and peroxisome. IDH1 is inactivated by Ser113phosphorylation and is expressed in many species including those withouta citric acid cycle. IDH1 appears to function normally as a tumorsuppressor which upon inactivation contributes to tumorigenesis partlythrough activation of the HIF-1 pathway (Bayley 2010; Reitman, 2010).Recent studies have implicated an inactivating mutation in IDH1 in theetiology of glioblasotoma (Bleeker, 2009; Bleeker, 2010).

Treatment with Coenzyme Q10 increased expression of IDH1 in cancer celllines including MCF-7, SKMEL28, HepG2 and PaCa-2 cells. There was amoderate increase in expression in the SCC25 cell lines. In contrastcultures of primary human derived fibroblasts HDFa, nFIB and the humanaortic smooth muscle cells HASMC did not demonstrate significant changesin the expression pattern of the IDH1 in response to Coenzyme Q10.α-ketoglutarate (α-KG) is a key intermediate in the TCA cycle,biochemically synthesized from isocitrate and is eventually converted tosuccinyl coA and is a druggable MIM and EpiShifter. The generation ofα-KG serves as a critical juncture in the TCA cycle as it can be used bythe cell to replenish intermediates of the cycle, resulting ingeneration of reducing equivalents to increase oxidativephosphorylation. Thus, Coenzyme Q10 mediated increase in IDH1 expressionwould result in formation of intermediates that can be used by themitochondrial TCA cycle to augment oxidative phosphorylation in cancercells. The results are summarized in the tables below.

TABLE 29 IDH1 in HDFa and MCF-7 Average Normalized Composition IntensityHDF, Media 346 HDF24-50-Coenzyme Q10 519 HDF24-100-Coenzyme Q10 600 MCF,Media 221 MCF24-50-Coenzyme Q10 336 MCF24-100-Coenzyme Q10 649

TABLE 30 IDH1 in HASMC vs. HepG2 after Treatment Normalized Amount -Composition Intensity HAS5g48-media 20 HAS5g48-50-Coenzyme Q10 948HAS5g48-100-Coenzyme Q10 1864 HAS22G48-Media 1917 HAS22G48-50-CoenzymeQ10 1370 HAS22G48-100-Coenzyme Q10 1023 Hep5g48-Media 14892Hep5g48-50-Coenzyme Q10 14106 Hep5g48-100-Coenzyme Q10 15774Hep22G48-Media 16558 Hep22G48-50-Coenzyme Q10 15537Hep22G48-100-Coenzyme Q10 27878

TABLE 31 IDH1 in HASMC vs. PACA2 after Treatment Amount - CompositionNormalized Intensity HAS5g48-media 562 HAS5g48-50-Coenzyme Q10 509HAS5g48-100-Coenzyme Q10 627 HAS22G48-Media 822 HAS22G48-50-Coenzyme Q101028 HAS22G48-100-Coenzyme Q10 1015 PACA5g48-Media 1095PACA5g48-50-Coenzyme Q10 1095 PACA5g48-100-Coenzyme Q10 860PACA22G48-Media 1103 PACA22G48-50-Coenzyme Q10 1503PACA22G48-100-Coenzyme Q10 1630

ATP Citrate Lyase (ACL)

ATP citrate Lyase (ACL) is a homotetramer (˜126 kd) enzyme thatcatalyzes the formation of acetyl-CoA and oxaloacetate in the cytosol.This reaction is a very important first step for the biosynthesis offatty acids, cholesterol, and acetylcholine, as well as for glucogenesis(Towle et al., 1997). Nutrients and hormones regulate the expressionlevel and phosphorylation status of this key enzyme. Ser454phosphorylation of ACL by Akt and PKA has been reported (Berwick, DC M Wet al., 2002; Pierce M W et al., 1982).

The data describes the effect of Coenzyme Q10 on ATP citrate Lyase isthat in normal and cancer cells. It is consistently observed that incancer cells there is a dose-dependent decrease in the expression of ACLenzymes. In contrast there appears to be a trend towards increasedexpression of ACL in normal cells. Cytosolic ACL has been demonstratedto be essential for histone acetylation in cells during growth factorstimulation and during differentiation. The fact that ACL utilizescytosolic glucose derived citrate to generate Acetyl CoA essential forhistone acetylation, a process important in the neoplastic processdemonstrates a role of Coenzyme Q10 induced ACL expression ininfluencing cancer cell function. Acetyl CoA generated from citrate bycytosolic ACL serves as a source for biosynthesis of new lipids andcholesterol during cell division. Thus, Coenzyme Q10 induced changes inACL expression alters Acetyl CoA availability for synthesis of lipidsand cholesterol in normal versus cancer cells. The results aresummarized in the tables below.

TABLE 32 ATPCL in HDFa and MCF-7 Composition Average NormalizedIntensity HDF-Media ~15000 HDF-50-Coenzyme Q10 ~17500 HDF-100-CoenzymeQ10 ~25000 MCF-Media ~7500 MCF-50-Coenzyme Q10 ~7500 MCF-100-CoenzymeQ10 ~12500

TABLE 33 ATP Citrate Lysase ~kd band in HASMC vs. HepG2 Amount -Composition Normalized Intensity HAS5g48-media 24557 HAS5g48-50-CoenzymeQ10 23341 HAS5g48-100-Coenzyme Q10 25544 HAS22G48-Media 27014HAS22G48-50-Coenzyme Q10 21439 HAS22G48-100-Coenzyme Q10 19491Hep5g48-Media 28377 Hep5g48-50-Coenzyme Q10 24106 Hep5g48-100-CoenzymeQ10 22463 Hep22G48-Media 24262 Hep22G48-50-Coenzyme Q10 31235Hep22G48-100-Coenzyme Q10 50588

TABLE 34 ATP Citrate Lysase ~kd band in HASMC vs. PACA2 Amount -Composition Normalized Intensity HAS5g48-media 11036 HAS5g48-50-CoenzymeQ10 12056 HAS5g48-100-Coenzyme Q10 15265 HAS22G48-Media 18270HAS22G48-50-Coenzyme Q10 15857 HAS22G48-100-Coenzyme Q10 13892PACA5g48-Media 11727 PACA5g48-50-Coenzyme Q10 8027 PACA5g48-100-CoenzymeQ10 4942 PACA22G48-Media 8541 PACA22G48-50-Coenzyme Q10 9537PACA22G48-100-Coenzyme Q10 14901

TABLE 35 ATP Citrate Lysase in HepG2 and PACA2 as % of CTRL Amount -Composition Normalized Intensity PACA5g48-Media 1.00PACA5g48-50-Coenzyme Q10 0.68 PACA5g48-100-Coenzyme Q10 0.42PACA22G48-Media 1.00 PACA22G48-50-Coenzyme Q10 1.12PACA22G48-100-Coenzyme Q10 1.74 Hep5g48-Media 1.00 Hep5g48-50-CoenzymeQ10 0.85 Hep5g48-100-Coenzyme Q10 0.79 Hep22G48-Media 1.00Hep22G48-50-Coenzyme Q10 1.29 Hep22G48-100-Coenzyme Q10 2.09

Pyruvate Kinase M2 (PKM2)

Pyruvate Kinase is an enzyme involved in the glycolytic pathway. It isresponsible for the transfer of phosphate from phosphoenolpyruvate (PEP)to adenosine diphosphophate (ADP) to generate ATP and pyruvate. PKM2 isan isoenzyme of the glycolytic pyruvate kinase, expression of which ischaracterized by the metabolic function of the tissue i.e. M2 isoenzymeis expressed in normal rapidly proliferating cells with high energyneeds such as embryonic cells and also expressed in few normaldifferentiated tissues such as lung and pancreatic islet cells thatrequire high rate of nucleic acid synthesis. PKM2 is highly expressed intumor cells due to their dependence on glycolytic pathway for meetingcellular energetic requirements. The PKM2 isoform normally thought to beembryonically restricted is re-expressed in cancerous cells. Cellsexpressing PKM2 favor a stronger aerobic glycolytic phenotype (show ashift in metabolic phenotype) with increased lactate production anddecreased oxidative phosphorylation. Thus, decrease in expression ofPKM2 in cancer cells would shift or down-regulate energy generation viathe glycolytic pathway, a strategy that is useful in the treatment ofcancer. Data demonstrates variable expression pattern of PKM2 in normaland cancer cells, with cancer cells demonstrating higher levels ofexpression compared to normal. Treatment of cells with Coenzyme Q10altered expression pattern of the PKM2 upper and lower band levels innormal and cancer cells. In cancer cells tested, there was adose-dependent decrease in the PKM2 expression, and no major changes innormal cells were observed. The results are summarized in the tablesbelow.

TABLE 36 Pyruvate Kinase Muscle form 2 Upper Band in HepG2 NormalizedVolume Normalized Intensity Amount - Composition (24 h) (48 h) 5g-Media28386 413 5g-50-Coenzyme Q10 29269 303 5g-100-Coenzyme Q10 18307 35422G-Media 25903 659 22G-50-Coenzyme Q10 22294 562 22G-100-Coenzyme Q1019560 601

TABLE 37 Pyruvate Kinase Muscle form 2 Lower Band (58 KD) in HepG2Normalized Volume Normalized Volume Amount - Composition (24 h) (48 h)5g-Media 10483 310 5g-50-Coenzyme Q10 11197 185 5g-100-Coenzyme Q10 7642122 22G-Media 9150 306 22G-50-Coenzyme Q10 6302 344 22G-100-Coenzyme Q106904 465

TABLE 38 Pyruvate Kinase Muscle form 2 Upper Band in HASMC Cells afterTreatment Amount - Composition Normalized Intensity 5g48-Media 6085g48-50-Coenzyme Q10 811 5g48-100-Coenzyme Q10 611 22G48-Media 51622G48-50-Coenzyme Q10 595 22G48-100-Coenzyme Q10 496 22G24-Media 30122G24-50-Coenzyme Q10 477 22G24-100-Coenzyme Q10 701

Lactate Dehydrogenase (LDH)

LDH is an enzyme that catalyzes the interconversion of pyruvate andlactate with the simultaneous interconversion of NADH and NAD⁺. It hasthe ability to convert pyruvate to lactate (lactic acid) under low celloxygen tension for generation of reducing equivalents and ATP generationat the expense of mitochondrial oxidative phosphorylation. Cancer cellstypically demonstrate increased expression of LDH to maintain theglycolytic flux to generate ATP and reducing equivalents and reducingmitochondrial OXPHOS. Thus, reducing the expression of the LDH in cancercells would shift metabolism from generation of lactate to facilitateentry of pyruvate into the TCA cycle. Treatment with Coenzyme Q10reduced Lactate Dehydrogenase (LDH) expression in cancer with minimaleffect on normal cells, supporting a role for Coenzyme Q10 in elicitinga shift in cancer cell bioenergetics for the generation of ATP fromglycolytic to mitochondrial OXPHOS sources by minimizing the conversionof cytoplasmic pyruvate to lactic acid. The results are summarized inthe tables below.

TABLE 39 Lactate Dehydrogenase in HepG2 Normalized Volume NormalizedVolume Amount - Composition (24 h) (48 h) 5g-Media 7981 59975g-50-Coenzyme Q10 7900 5188 5g-100-Coenzyme Q10 6616 7319 22G-Media9171 7527 22G-50-Coenzyme Q10 7550 6173 22G-100-Coenzyme Q10 7124 9141

TABLE 40 Lactate Dehydrogenase in HepG2 as % Control from 2 ExperimentsAverage Volume as a Amount - Composition % of Control 5g24-Media 1.005g24-50-Coenzyme Q10 0.64 5g24-100-Coenzyme Q10 1.06 5g48-Media 1.005g48-50-Coenzyme Q10 1.12 5g48-100-Coenzyme Q10 1.21 22G24-Media 1.0022G24-50-Coenzyme Q10 1.21 22G24-100-Coenzyme Q10 1.44 22G48-Media 1.0022G48-50-Coenzyme Q10 0.95 22G48-100-Coenzyme Q10 0.67

TABLE 41 Lactate Dehydrogenase in PACA2 Normalized Volume NormalizedVolume Amount - Composition (24 h) (48 h) 5g-Media 2122 23605g-50-Coenzyme Q10 5068 2978 5g-100-Coenzyme Q10 3675 2396 22G-Media4499 2332 22G-50-Coenzyme Q10 10218 2575 22G-100-Coenzyme Q10 7158 3557

Pyruvate Dehydrogenase—B (PDH-E1)

Pyruvate Dehydrogenase beta (PDH-E1) is the first enzyme component thatis part of the pyruvate dehydrogenase complex (PDC) that convertspyruvate to acetyl CoA. PDH-E1 requires thiamine as cofactor for itsactivity, performs the first two biochemical reactions in the PDCcomplex essential for the conversion of pyruvate to acetyl CoA to enterthe TCA cycle in the mitochondria. Thus, concomitant decreases in PKM2and LDH expression along with increase in expression of PDH-E1 in cancercells would enhance the rate of entry of pyruvate towards augmenting themitochondrial OXPHOS for generation of ATP. The data shows that forexpression of PDH-E1 in normal and cancer cell lines, the baselineexpressions of this enzyme is decreased in cancer compared to normalcells. Treatment with Coenzyme Q10 is associated with progressiveincrease in the expression of the PDH-E1 proteins in cancer cells withminimal changes in the normal cells. The results are summarized in thetables below.

TABLE 42 Pyruvate Dehydrogenase Beta in HepG2 Normalized VolumeNormalized Volume Amount - Composition (24 h) (48 h) 5g-Media 517 1005g-50-Coenzyme Q10 921 123 5g-100-Coenzyme Q10 433 205 22G-Media 484 18122G-50-Coenzyme Q10 426 232 22G-100-Coenzyme Q10 340 456

TABLE 43 Pyruvate Dehydrogenase Beta in PACA2 Normalized VolumeNormalized Volume Amount - Composition (24 h) (48 h) 5g-Media 323 3755g-50-Coenzyme Q10 492 339 5g-100-Coenzyme Q10 467 252 22G-Media 572 27622G-50-Coenzyme Q10 924 279 22G-100-Coenzyme Q10 1201 385

TABLE 44 Pyruvate Dehydrogenase Beta in HASMC after Treatment Amount -Composition Normalized Volume 5g48-Media 140 5g48-50-Coenzyme Q10 1475g48-100-Coenzyme Q10 147 22G48-Media 174 22G48-50-Coenzyme Q10 14922G48-100-Coenzyme Q10 123 22G24-Media 140 22G24-50-Coenzyme Q10 14522G24-100-Coenzyme Q10 150

Caspase 3

Control of the onset of apoptosis is often exerted at the level of theinitiator caspases, caspase-2, -9 and -8/10. In the extrinsic pathway ofapoptosis, caspase-8, once active, directly cleaves and activatesexecutioner caspases (such as caspase-3). The active caspase-3 cleavesand activates other caspases (6, 7, and 9) as well as relevant targetsin the cells (e.g. PARP and DFF). In these studies, the levels ofeffectors caspase-3 protein were measured in the cancer cell lines andin normal cell lines in response to Coenzyme Q10. It should be notedalthough control of apoptosis is through initiator caspases, a number ofsignaling pathways interrupt instead the transmission of the apoptoticsignal through direct inhibition of effectors caspases. For e.g. P38MAPK phosphorylates caspase-3 and suppresses its activity(Alvarado-Kristensson et al., 2004). Interestingly, activation ofprotein phosphates (PP2A) in the same study or protein kinase C delta(PKC delta) (Voss et al., 2005) can counteract the effect of p38 MAPK toamplify the caspase-3 activation and bolster the transmission of theapoptotic signal. Therefore, events at the level of caspase-3 activationor after Caspase 3 activation may determine the ultimate fate of thecell in some cases.

Caspase-3 is a cysteine-aspartic acid protease that plays a central rolein the execution phase of cell apoptosis. The levels of caspase 3 in thecancer cells were increased with Coenzyme Q10 treatment. In contrast theexpression of Caspase-3 in normal cells was moderately decreased innormal cells. The results are summarized in the tables below.

TABLE 45 Caspase 3 in PACA2 Normalized Volume Normalized VolumeAmount-Composition (24 h) (48 h) 5g-Media 324 300 5g-50-Coenzyme Q10 325701 5g-100-Coenzyme Q10 374 291 22G-Media 344 135 22G-50-Coenzyme Q10675 497 22G-100-Coenzyme Q10 842 559

TABLE 46 Caspase 3 in HepG2 cells as % Control from 2 ExperimentsNormalized Volume as Amount-Composition a % of Control 5g24-Media 1.005g24-50-Coenzyme Q10 1.08 5g24-100-Coenzyme Q10 1.76 5g48-Media 1.005g48-50-Coenzyme Q10 1.44 5g48-100-Coenzyme Q10 0.95 22G24-Media 1.0022G24-50-Coenzyme Q10 1.39 22G24-100-Coenzyme Q10 1.78 22G48-Media 1.0022G48-50-Coenzyme Q10 1.50 22G48-100-Coenzyme Q10 1.45

TABLE 47 Caspase 3 in HASMC after Treatment Amount-CompositionNormalized Volume 5g48-Media 658 5g48-50-Coenzyme Q10 7665g48-100-Coenzyme Q10 669 22G48-Media 846 22G48-50-Coenzyme Q10 63922G48-100-Coenzyme Q10 624 22G24-Media 982 22G24-50-Coenzyme Q10 83522G24-100-Coenzyme Q10 865

Succinate Dehydrogenase (SDH)

Succinate dehydrogenase, also known as succinate-coenzyme Q reductase isa complex of the inner mitochondrial membrane that is involved in bothTCA and electron transport chain. In the TCA, this complex catalyzes theoxidation of succinate to fumarate with the concomitant reduction ofubiquinone to ubiquinol. (Baysal et al., Science 2000; and Tomlinson etal., Nature Genetics 2002). Germline mutations in SDH B, C and Dsubunits were found to be initiating events of familial paraganglioma orleiomyoma (Baysal et al., Science 2000).

Western blotting analysis was used to characterize expression of SDHSubunit B in mitochondrial preparations of cancer cells treated withCoenzyme Q10. The results suggest that Coenzyme Q10 treatment isassociated with increase SDH protein levels in the mitochondrion of thecells. These results suggest one of the mechanisms of action of CoenzymeQ10 is to shift the metabolism of the cell towards the TCA cycle and themitochondrion by increasing the levels of mitochondrial enzymes such asSDHB. The results are summarized in the table below.

TABLE 48 Succinate Dehydrogenase B in NCIE0808 Mitopreps AverageNormalized Composition-Time Volume Media 531  50 uM Coezyme Q10, 3 h 634100 uM Coenzyme Q10, 3 h 964  50 uM Coenzyme Q10, 6 h 1077 100 uMCoenzyme Q10, 6 h 934

Hypoxia Induced Factor-1

Hypoxia inducible factor (Hif) is a transcription factor composed ofalpha and beta subunits. Under normoxia, the protein levels of Hif1alpha are very low owing to its continuous degradation via a sequence ofpost translational events. The shift between glycolytic and oxidativephosphorylation is generally considered to be controlled by the relativeactivities of two enzymes PDH and LDH that determine the catabolic fateof pyruvate. Hif controls this crucial bifurgation point by inducing LDHlevels and inhibiting PDH activity by stimulating PDK. Due to thisability to divert pyruvate metabolism from mitochondrion to cytosol, Hifis considered a crucial mediator of the bioenergetic switch in cancercells.

Treatment with Coenzyme Q10 decreased Hif1 alpha protein levels after inmitochondrial preparations of cancer cells. In whole cell lysates ofnormal cells, the lower band of Hif1a was observed and showed a decreaseas well. The results are summarized in the tables below.

TABLE 49 Hif1 alpha Lower Band in HASMC Cells after TreatmentAmount-Composition Normalized Volume 5g48-Media 22244 5g48-50-CoenzymeQ10 21664 5g48-100-Coenzyme Q10 19540 22G48-Media 1475222G48-50-Coenzyme Q10 17496 22G48-100-Coenzyme Q10 23111 22G24-Media21073 22G24-50-Coenzyme Q10 18486 22G24-100-Coenzyme Q10 17919

TABLE 50 Hif1 alpha Upper Band in HepG2 after TreatmentAmount-Composition Normalized Volume 5g24-Media 12186 5g24-50-CoenzymeQ10 8998 5g24-100-Coenzyme Q10 9315 5g48-Media 8868 5g48-50-Coenzyme Q108601 5g48-100-Coenzyme Q10 10192 22G24-Media 11748 22G24-50-Coenzyme Q1014089 22G24-100-Coenzyme Q10 8530 22G48-Media 8695 22G48-50-Coenzyme Q109416 22G48-100-Coenzyme Q10 5608

Example 21 Analysis of Oxygen Consumption Rates (OCR) and ExtracellularAcidification (ECAR) in Normal and Cancer Cells Treated with CoQ10

This example demonstrates that exposure of cells to treatment by arepresentative MIM/epi-shifter of the invention—CoQ10—in the absenceand/or presence of stressors (e.g., hyperglycemia, hypoxia, lacticacid), is associated with a shift towards glycolysis/lactatebiosynthesis and mitochondrial oxidative phosphorylation (as measured byECAR and OCR values) representative of values observed in a normal cellsunder normal physiological conditions.

Applicants have demonstrated in the previous section that treatment withCoQ10 in cancer cells is associated with changes in expression ofspecific proteins that enhance mitochondrial oxidative phosphorylation,with a concomitant decrease in glycolysis and lactate biosynthesis. Thisexample shows that a direct measure of mitochondrial oxidativephosphorylation can be obtained by measuring the oxygen consumptionrates (OCR) in cell lines using the SeaHorse XF analyzer, an instrumentthat measures dissolved oxygen and extracellular pH levels in an invitro experimental model. (SeaHorse Biosciences Inc, North Billerica,Mass.).

The pH of the extracellular microenvironment is relatively acidic intumors compared to the intracellular (cytoplasmic) pH and surroundingnormal tissues. This characteristic of tumors serves multiple purposes,including the ability to invade the extracellular matrix (ECM), ahallmark attribute of tumor metastasis that subsequently initiatessignaling cascades that further modulate:

-   -   tumor angiogenesis    -   increased activation of arrest mechanisms that control cell        cycle turn-over    -   immuno-modulatory mechanisms that facilitate a cellular evasion        system against immunosurveillance    -   metabolic control elements that increase dependency on        glycolytic flux and lactate utilization    -   dysregulation of key apopototic gene families such as Bcl-2,        IAP, EndoG, AIF that serve to increase oncogenicity

While not wishing to be bound by any particular theory, the acidic pH ofthe external microenvironment in the tumor is a consequence of increasein hydrogen ion concentrations extruded from the tumor cells due to theincreased lactate production from an altered glycolytic phenotype.

In this experiment, the OCR and extracellular acidification rate (ECAR)in normal cells lines were obtained in the presence and absence of CoQ10to determine baseline values. It was observed that in its nativenutrient environment, the basal OCR rates in normal cells lines aredifferent, and are usually a function of the physiological roles of thecells in the body.

For example, one set of experiments were conducted using thenon-cancerous cell line HDFa, which is a human adult dermal fibroblastcell line. Fibroblasts are cells that primarily synthesize and secreteextracellular matrix (ECM) components and collagen that form thestructural framework (stroma) for tissues. In addition, fibroblasts areknown to serve as tissue ambassadors of numerous functions such as woundhealing and localized immunomodulation. Under normal physiologicalconditions, energy requirements in normal fibroblasts are met using acombination of glycolysis and oxidative phosphorylation—the glycolysisproviding the necessary nutrients for synthesis of ECM.

In contrast to HDFa, the HASMC (human aortic smooth muscle cell) isfound in arteries, veins, lymphatic vessels, gastrointestinal tracts,respiratory tract, urinary bladder and other tissues with the ability toundergo regulated excitation-contraction coupling. The ability of smoothmuscles such as HASMC cells to undergo contraction requires energyprovided by ATP. These tissues transition from low energy modes whereinATP may be supplied from mitochondria to high energy modes (duringexercise/stress) where energy is provided by switching to glycolysis forrapid generation of ATP. Thus, normal smooth muscle cells can use acombination of mitochondrial OXPHOS and glycolysis to meet their energyrequirements under normal physiological environment.

The differences in their respective physiological roles (i.e., HDFa andHASMC) were observed in the resting OCR values measured in these cellslines using the SeaHorse XF analyzer. FIGS. 29 and 30 describe the OCRin HDFa and HASMC cells grown in physiologically normal glucose (about4.6 mM) and high glucose (hyperglycemic) conditions.

The baseline OCR values for HDFa in the absence of any treatments undernormal oxygen availability is approximately 40 pmoles/min (FIG. 29) inthe presence of 5.5 mM glucose. This value was slightly elevated whenthe cells were maintained at 22 mM glucose. In contrast, in HASMC cells,the OCR values at 5.5 mM glucose is approximately 90 pmoles/min, and theOCR value declined to approximately 40 pmoles/min while at 22 mMglucose. Thus, under hyperglycemic conditions, there is a differentialresponse between HDFa and HASMC, further demonstrating inherentdifferences in their respective physiological make-up and function.

Treatment with CoQ10 in cells is associated with changes in OCR that isrepresentative of conditions observed at normal (5 mM) glucoseconditions. The complexity of physiological response is compounded inthe presence of low oxygen tension. Thus, CoQ10 exposure is associatedwith changes in OCR rates in normal cells towards a physiological statethat is native to a particular cell.

Table 51 below describes the ECAR values (mpH/min) in HDFa cells in thepresence or absence of CoQ10 under normoxic and hypoxic conditions at5.5 mM and 22 mM glucose. It can be observed that in normal cells,treatment with CoQ10 had minimal influence on ECAR values, even thoughit influenced OCR in these cells. In high glucose hypoxic conditions,treatment with CoQ10 was associated with lowering of elevated ECAR to avalue that was observed in untreated normoxic conditions.

TABLE 51 ECAR values in HDFa cells in the absence and presence of CoQ10under normoxic and hypoxic conditions at 5.5 mM and 22 mM glucoseNormoxia Hypoxia Normoxia Hypoxia (5.5 mM) (5.5 mM) (22 mM) (22 mM)Treatment ECAR SEM ECAR SEM ECAR SEM ECAR SEM Untreated 5 1.32 5 0.62 50.62 9 0.81  50 μM 6 1.11 5 0.78 5 0.78 6 0.70 31510 100 μM 6 0.76 51.19 5 1.19 8 1.07 31510

In Table 52 the measured baseline ECAR values (mpH/min) in HASMC werehigher compared to that of HDFa. Induction of hypoxic conditions causedan increase in ECAR most likely associated with intracellular hypoxiainduced acidosis secondary to increased glycolysis.

TABLE 52 ECAR values in HASMC cells in the absence and presence of CoQ10under normoxic and hypoxic conditions at 5.5 mM and 22 mM glucoseNormoxic Hypoxic Normoxic Hypoxic (5.5 mM) (5.5 mM) (22 mM) (22 mM)Treatment ECAR SEM ECAR SEM ECAR SEM ECAR SEM Untreated 9 2.22 11 2.1822 2.08 19 1.45  50 μM 9 2.13 11 2.54 21 1.72 17 1.60 31510 100 μM 91.72 13 2.30 22 1.64 17 1.47 31510

Treatment with CoQ10 was observed to be associated with a downward trendof ECAR rates in hyperglycemic HASMC cells in hypoxic conditions towardsa value that would be observed in normoxic normal glucose conditions.These data demonstrate the presence of physiological variables that isinherent to the physiological role of a specific type of cell,alterations observed in abnormal conditions (e.g. hyperglycemia) isshifted towards normal when treated with CoQ10.

In contrast, cancer cells (e.g., MCF-7, PaCa-2) are inherently primed toculture at higher levels of glucose compared to normal cells due totheir glycolytic phenotype for maintenance in culture. Treatment withCoQ10 caused a consistent reduction in OCR values (FIG. 31 and FIG. 32).

The effects of CoQ10 on OCR values in MCF-7 and PaCa-2 cells was similarto that of the normal HDFa and HASMC cells, wherein the variableresponse was suggestive of a therapeutic response based on individualmetabolic profile of the cancer cell line.

TABLE 53 ECAR values in PaCa-2 cells in the absence and presence ofCoQ10 under normoxic and hypoxic conditions at 5.5 mM and 22 mM glucoseNormoxia Hypoxia Normoxia Hypoxia (17 mM) (17 mM) (22 mM) (22 mM)Treatment ECAR SEM ECAR SEM ECAR SEM ECAR SEM Untreated 21 5.97 16 3.4124 4.35 36 5.65  50 μM 13 3.08 12 1.66 20 5.15 25 4.58 31510 100 μM 142.14 17 2.59 19 3.38 30 5.62 31510

Table 53 describes the ECAR values in PaCa-2 cells. In contrast tonormal cells, cancer cells are phenotypically primed to use high glucosefor ATP generation (enhanced glycolysis) resulting in higher ECAR (Table53, ECAR for untreated normoxia 17 mM) at 21 mpH/min. Treatment withCoQ10 produces a significant decrease in ECAR rates under theseconditions, most likely associated with a decrease in the glycolysisgenerated lactic acid. The associated decrease in OCR in these cells waslikely associated with increased efficiency of the mitochondrial OXPHOS.

A similar comparison of OCR and ECAR values (data not shown) weredetermined in numerous other normal and cancer cells lines, including:HAEC (normal human aortic endothelial cells), MCF-7 (breast cancer),HepG2 (liver cancer) and highly metastatic PC-3 (prostate cancer) celllines. In all of the cell lines tested, exposure to CoQ10 in the absenceand/or presence of stressors (e.g., hyperglycemia, hypoxia, lactic acid)was associated with a shift in OCR and ECAR values representative ofvalues observed in a normal cells under normal physiological conditions.Thus, the overall effect of CoQ10 in the treatment of cancer, includingcell death, is an downstream effect of its collective influence onproteomic, genomic, metabolomic outcomes in concert with shifting of thecellular bioenergetics from glycolysis to mitochondrial OXPHOS.

Example 22 Building Block Molecules for the Biosynthesis of CoQ10

This example demonstrates that certain precursors of CoQ10 biosynthesis,such as those for the biosynthesis of the benzoquinone ring, and thosefor the biosynthesis of the isoprenoid repeats and their attachment tothe benzoquinone ring (“building block components”), can be individuallyadministered or administered in combination to target cells, and effectdown-regulation of the apoptosis inhibitor Bcl-2, and/or up-regulationof the apoptosis promoter Caspase-3. Certain precursors or combinationsthereof may also inhibit cell proliferation. The data suggests that suchCoQ10 precursors may be used in place of CoQ10 to achieve substantiallythe same results as CoQ10 administration.

Certain exemplary experimental conditions used in the experiments arelisted below.

Skmel-28 melanoma cells were cultured in DMEM/F12 supplemented with 5%Fetal Bovine Serum (FBS) and 1× final concentration of Antibiotics. Thecells were grown to 85% confluency and treated with building blockcomponents for 3, 6, 12 and 24 hours. The cells were then pelleted and aWestern blot analysis was performed.

The test building block components included L-Phenylylalanine,DL-Phenylyalanine, D-Phenylylalanine, L-Tyrosine, DL-Tyrosine,D-Tyrosine, 4-Hydroxy-phenylpyruvate, phenylacetate,3-methoxy-4-hydroxymandelate (vanillylmandelate or VMA), vanillic acid,4-hydroxy-benzoate, pyridoxine, panthenol, mevalonic acid,Acetylglycine, Acetyl-CoA, Farnesyl, and2,3-Dimethoxy-5-methyl-p-benzoquinone.

In the Western Blot Analysis, the cells were pelleted in cold PBS,lysed, and the protein levels were quantified using a BCA protein assay.The whole cell lysate was loaded in a 4% loading 12% running Tris-HClgel. The proteins were then transferred to a nitrocellulose paper thenblocked with a 5% milk Tris-buffered solution for 1 hour. The proteinswere then exposed to primary antibodies (Bcl-2 and Caspase-3) overnight.The nitrocellulose paper was then exposed to Pico Chemilluminescent for5 min and the protein expression was recorded. After exposure, actin wasquantified using the same method. Using ImageJ the levels of proteinexpression were quantified. A t-Test was used to analyze for statisticalsignificance.

Illustrative results of the experiments are summarized below.

Western Blot Analysis of Building Block component L-Phenylalanine:Before proceeding to the synthesis pathway for the quinone ringstructure, L-Phenylalanine is converted to tyrosine. A western blotanalysis was performed to quantify any changes in the expression of theapoptotic proteins in the melanoma cells. The concentrations tested were5 μM, 25 μM, and 100 μM. Initial studies added L-Phenylalanine toDMEM/F12 medium which contained a concentration of 0.4 M phenylalanine.For the 5 μM, 25 μM, and 100 μM the final concentration of theL-Phenylalanine in the medium was 0.405 M, 0.425 M, and 0.500 M,respectively. These final concentrations were tested on the Skmel-28cells for incubation periods of 3, 6, 12 and 24 hours. The cells weregrown to 80% confluency before adding the treatment medium and harvestedusing the western blot analysis procedure as described above. Astatistically significant decrease in Bcl-2 was observed for the 100 μML-Phenylalanine after 3 hours and 12 hours incubation. Fr the 5 μML-phenylalanine, a statistically significant decrease in Bcl-2 wasobserved after 6 hours of incubation. For the 25 μM L-phenylalanine, astatistically significant decrease in Bcl-2 and a statisticallysignificant increase in Caspase-3 were observed after 12 hours ofincubation. A statistically significant decrease in Bcl-2 indicates achange in the apoptotic potential and a statistically significantincrease in Caspase-3 confirms the cells are undergoing apoptosis. Therewas a constant trend for the decrease in Bcl-2 compared to the controleven though, due to sample size and standard deviation, these timepoints were not statistically significant in this experiment.

Western Blot Analysis of Building Block component D-Phenylalanine:D-Phenylalanine, a chemically synthetic form of the bioactiveL-Phenylalanine, was tested for comparison to L-phenylalanine. For allthree concentrations (5 μM, 25 μM, and 100 μM of D-Phenylalanine, therewas a significant reduction in Bcl-2 expression after 6 hours ofincubation. In addition, for the 5 μM and 25 μM, there was a significantreduction after 3 hours of incubation. For the 5 μM and 100 μMconcentrations, a significant increase in Caspase-3 expression wasobserved after 6 hours of incubation.

Western Blot Analysis of Building Block component DL-Phenylalanine:DL-Phenylalanine was also tested for comparison to L-Phenylalanine.Again, concentrations of 5 μM, 25 μM, and 100 μM were tested on Skmel-28cells. The incubation periods were 3, 6, 12 and 24 hours. Astatistically significant increase in Caspase-3 was observed after 3hours of incubation. A statistically significant decrease in Bcl-2 wasobserved after 24 hours of incubation. Although a decreasing Bcl-2 andincreasing Caspase-3 trend at all other concentrations and incubationtime points, they were not statistically significant in this experiment.

Western Blot Analysis of Building Block component L-Tyrosine: L-Tyrosineis a building block component for the synthesis of quinone ringstructure of CoQ10. Initial testing of L-Tyrosine did not result in ahigh enough protein concentration for western blot analysis. From thisstudy concentrations under 25 μM were tested for Western Blot Analysis.The DMEM/F12 medium used contained L-Tyrosine disodium saltconcentration of 0.398467 M. The initial concentration was increased by500 nM, 5 μM, and 15 μM. A statistically significant increase inCaspase-3 was observed for the 500 nM concentration after 12 hours ofincubation. A statistically significant increase in Caspase-3 was alsoobserved for the 5A statistically significant decrease in Bcl-2 wasobserved for the 5 μM concentration after 24 hours of incubation. Astatistically significant decrease in Bcl-2 was observed for the 500 μMand 5 μM concentrations after 24 hours of incubation.

Western Blot Analysis of Building Block component D-Tyrosine:D-Tyrosine, a synthetic form of L-Tyrosine, was tested for comparisonagainst the L-Tyrosine apoptotic effect on the melanonal cells. Based oninitial studies with L-Tyrosine, concentrations below 25 μM were chosenfor the western blot analysis. The concentrations tested were 1 μm, 5μM, and 15 μM. D-Tyrosine showed a reduction in Bcl-2 expression for the5 μM and 15 μM concentrations for 12 and 24 hour time periods. Caspase-3was significantly increased for the concentration of 5 μM for 3, 12 and24 time periods. Also there was an increase in Caspase-3 expression forthe 1 μM for 12 and 24 hour time period. In addition there is anincrease in Caspase-3 expression for 5 μM for the 12 hour time period.

Western Blot Analysis of Building Block component DL-Tyrosine:DL-Tyrosine, a synthetic form of L-Tyrosine, was also tested forcomparison against L-Tyrosine's apoptotic effect on the cells. There isa statistical decrease in Bcl-2 expression seen in the 1 μM and 15 μMconcentrations after 12 hours incubation and for the 5 μM after 24 hourof incubation. An increase in Caspase-3 expression was also observed forthe 5 μM and 15 μM after 12 hours of incubation.

Western Blot Analysis of Building Block component4-Hydroxy-phenylpyruvate: 4-Hydroxy-phenylpyruvate is derived fromTyrosine and Phenylalanine amino acids and may play a role in thesynthesis of the ring structure. The concentration of 1 μM, 5 μM, and 15μM were tested for Bcl-2 and Caspase-3 expression. For the 5 μM and 15μM concentrations there is a significant reduction in Bcl-2 expressionafter 24 hours of incubation and a significant increase in Caspase-3expression after 12 hours of incubation.

Western Blot Analysis of Building Block component Phenylacetate:Phenylacetate has the potential to be converted to 4-Hydroxy-benzoate,which plays a role in the attachment of the side chain to the ringstructure. The concentration tested were 1 μM, 5 μM, and 15 μM. Forphenylacetate there was a decrease in Bcl-2 expression for theconcentration of 5 μM and 15 μM after 12 hours and 24 hours ofincubation. An increase in Caspase-3 expression was observed for theconcentration of 5 μM and 15 μM after 12 hours and 24 hours ofincubation.

Western Blot Analysis of Building Block component3-methoxy-4-hydroxymandelate (vanillylmandelate or VMA): VMA is anadditional component for the synthesis of the CoQ10 quinone ringstructure. The concentrations tested were 100 nM, 250 nM, 500 nM, 1 μM,25 μM, 50 μM, and 100 μM. Though no statistically significant apoptoticeffect was observed in this experiment, the data indicated a downwardtrend of Bcl-2 expression.

Western Blot Analysis of Building Block component Vanillic acid:Vanillic is a precursor for the synthesis of the quinone ring and wastested at a concentration of 500 nm, 5 μM, and 15 μM. A western blotanalysis measured Bcl-2 and Caspase-3 expression. Vanillic Acid wasshown to significantly reduce Bcl-2 expression for the concentrations of500 nM and 5 μM at the 24 hour incubation time point. For the 15 μMconcentration there is a reduction in Bcl-2 expression after 3 hours ofincubation. For the cells incubated with 15 μM for 24 hours there was asignificant increase in Caspase-3 expression.

Western Blot Analysis of Building Block component 4-Hydroxybenzoate:4-Hydroxybenzoate acid plays a role in the attachment of the isoprenoidside chain to the ring structure. The concentrations tested were 500 nM,1 μM, and 50 μM. There was a significant reduction in Bcl-2 expressionfor the 15 μM concentration after 24 hours of incubation.

Western Blot Analysis of Building Block component 4-Pyridoxine:Pyridoxine is another precursor building block for the synthesis of thequinone ring structure of CoQ10. The concentrations tested for thiscompound are 5 μM, 25 μM, and 100 μM. The cells were assayed for theirlevels of Bcl-2 and Caspase-3. Pyridoxine showed a significant reductionin Bcl-2 after 24 hours of incubation in melanoma cells.

Western Blot Analysis of Building Block component Panthenol: Panthenolplays a role in the synthesis of the quinone ring structure of CoQ10.The concentrations tested on melanoma cells were 5 μM, 25 μM, and 100μM. This compound showed a significant reduction in Bc1-2 expression forthe 25 μM concentration.

Western Blot Analysis of Building Block component Mevalonic: MevalonicAcid is one of the main components for the synthesis of CoQ10. Thiscompound was tested at the concentrations of 500 nM, 1 μM, 25 μm, and 50μM. There was no significant reduction in Bcl-2 expression or anincrease in Caspase-3 expression in this experiment.

Western Blot Analysis of Building Block component Acetylglycine: Anotherroute for the synthesis of CoQ10 is the isoprenoid (side chain)synthesis. The addition of Acetylglycine converts Coenzyme A toAcetyl-CoA which enters the mevalonic pathway for the synthesis of theisoprenoid synthesis. The concentrations tested were 5 μM, 25 μM, and100 μM. The testing of Acetylglycine showed significant decrease inBcl-2 expression after 12 hours of incubation for the concentration of 5μM and 25 μM. A significant decrease in Bcl-2 was recorded for the 100μM concentration at the 24 hour incubation time point.

Western Blot Analysis of Building Block component Acetyl-CoA: Acetyl-CoAis a precursor for the mevalonic pathway for the synthesis of CoQ10. Theconcentrations tested were 500 nm, 1 μM, 25 μM, and 50 μM. There was nosignificant observed reduction in Bcl-2 or increase in Caspase-3expression for the time points and concentrations tested.

Western Blot Analysis of Building Block component L-Tyrosine incombination with farnesyl: L-Tyrosine is one of the precursors for thesynthesis of the quinone ring structure for CoQ10. Previous experimenttested the reaction of L-Tyrosine in medium with L-Phenylalanine andL-Tyrosine. In this study L-Tyrosine was examined in medium without theaddition of L-Phenylalanine and L-Tyrosine. In this study the finalconcentrations of L-Tyrosine tested were 500 nM, 5 μM, and 15 μM.Farnesyl was tested at a concentration of 50 μM. There was no observedsignificant response for the 3 and 6 hour time points.

Western Blot Analysis of Building Block component L-Phenylalanine incombination with Farnesyl: L-Phenylalanine, a precursor for thesynthesis of the quinone ring structure, was examine in combination withfarnesyl in medium free of L-Tyrosine and L-Phenylalanine. A westernblot analysis was performed to assay the expression of Bcl-2 andCaspase-3. The final concentrations of L-Phenylalanine were: 5 μM, 25μM, and 100 μM. Farnesyl was added at a concentration of 50 μM. Thisstudy showed a decrease in Bc1-2 expression for most of theconcentrations and combinations tested as depicted in the table below.

TABLE 54 L-Phenylalanine and/or Farnesyl L-Phenyl- 3 hr 6 hr 12 hr 24 hralanine Bcl-2 Cas-3 Bcl-2 Cas-3 Bcl-2 Cas-3 Bcl-2 Cas-3  5 μM X  5 μM XX w/Farnesyl  25 μM X X  25 μM X X w/Farnesyl 100 μM X X X 100 μM Xw/Farnesyl

Cell Proliferation Assay of the Combination of 4-Hydroxy-Benzoate withBenzoquinone: This set of experiments used a cell proliferation assay toassess the effect of combining different building block molecules oncell proliferation.

The first study examined the effect of combining 4-Hydroxy-Benzoate withBenzoquinone. Cells were incubated for 48 hours, after which a cellcount was performed for the live cells. Each test group was compared tothe control, and each combination groups were compared to Benzoquinonecontrol. The compounds were statistically analyzed for the addition ofBenzoquinone. The following table summarizes the cell count resultswherein the X mark indicates a statistical decrease in cell number.

TABLE 55 4-Hydroxy-Benzoate and/or Benzoquinone Compared to 4- Hydroxyto Compared to Compared compound w/o Benzoquinone 4-Hydroxy to CtrlBenzoquinone Control 500 nm X 500 nm w/Benzo X X (35 μM) 500 nm w/BenzoX X (70 μM)  1 μm X  1 μm w/Benzo X X (35 μM)  1 μm w/Benzo X X (70 μM) 50 μm X  50 μm w/Benzo X (35 μM)  50 μm w/Benzo X X X (70 μM)

There is a significant decrease in cell number for the cells incubatedwith 4-Hydroxybenzoic and benzoquinone and in combination. For thecombination of 50 μM 4-Hydroxybenzoate in combination with 70 μMBenzoquinone there is significant reduction in cell number compared tothe Benzoquinone control. This suggests a synergistic effect for thismolar ratio.

Additional studies were performed testing additional molar ratios. Forthe first test 4-Hydroxybenzoic were tested at concentrations of 500 nM,1 μM, and 50 μM. These concentrations were tested in combination with2,3-Dimethoxy-5-methyl-p-benzoquinone (Benzo). The concentration ofBenzo tested were 25 μM, 50 μM, and 100 μM. Melanoma cells were grown to80% confluency and seeded in 6 well plates at a concentration of 40Kcells per well. The cells were treated with CoQ10, 4-Hydroxybenzoate,Benzo, and a combination of 4-Hydroxybenzoate/Benzo.

A T-test was performed with p<0.05 as statistically significant. An Xsignifies a statistical decrease in cell number.

TABLE 56 4-Hydroxybenzoic and/or 2,3-Dimethoxy-5- methyl-p-benzoquinone(Benzo) Ctrl vs Benzo 25 μM X Ctrl vs Benzo (B) 50 μM Ctrl vs Benzo (B)100 μM X Ctrl vs 4-Hydroxybenzoate (HB) 500 nm X Ctrl vs HB 1 μM X Ctrlvs HB 50 μM X 500 nM HB vs 500 nM HB w/25 B X 500 nM HB vs 500 nM HBw/50 B X 500 nM HB vs 500 nM HB w/100 B X 1 uM HB vs 1 μM HB w/25 B X 1uM HB vs 1 μM HB w/50 B X 1 uM HB vs 1 μM HB w/100 B 50 uM HB vs 50 μMHB w/25 B X 50 uM HB vs 50 μM HB w/50 B X 50 uM HB vs 50 μM HB w/100 B500 nM HB w/25 B vs 25 B X 500 nM HB w/50 B vs 50 B X 500 nM HB w/100 Bvs 100 B X 1 μM HB w/25 B vs 25 B X 1 μM HB w/50 B vs 50 B X 1 μM HBw/100 B vs 100 B 50 μM HB w/25 B vs 25 B X 50 μM HB w/50 B vs 50 B X 50μM HB w/100 B vs 100 B

There is a significant decrease in cell proliferation for the treatmentmedium containing HB. Moreover the combination of the HB withbenzoquinone showed a significant reduction in cell number compare tothe cells incubated with the corresponding benzoquinone concentrations.

A cell proliferation assay was also performed on neonatal fibroblastcells. The concentrations of HB tested were 500 nM, 5 μM, and 25 μM. HBwas also tested in combination with benzoquinone at a concentrations of25 μM, 50 μM, and 100 μM. Melanoma cells were seeded at 40 k cells perwell and were treated for 24 hours. The cells were trypsinized andquantified using a coulter counter.

Statistical analysis did not show a significant reduction in fibroblastcells. This indicates minimal to no toxicity in normal cells.

Cell Proliferation Assay of the Combination of phenylacetate andbenzoquinone: Phenyl acetate is a precursor for the synthesis of4-Hydroxybenzoic acid (facilitates the attachment of the ring structure.A cell proliferation assay was performed to assay the effect ofincubating phenylacetate in combination with CoQ10 and Benzoquinone.

TABLE 57 Phenylacetate and/or Benzoquinone Ctrl and 25/25 μM Ben X Ctrland 25/50 μM Ben X Ctrl and 25/100 μM Ben X Ctrl and 25/25 μM Q-10 XCtrl and 25/25 μM Q-10 X Ctrl and 25/50 μM Q-10 X Ctrl and 25/100 μMQ-10 X Ctrl and Ben 25 X Ctrl and Ben 50 X Ctrl and Ben 100 X Ctrl andQ-10 25 Ctrl and Q-10 50 Ctrl and Q-10 100 X Ben 25 μM and 500 nM/25 μMBen X Ben 25 μM and 5 nM/25 μM Ben X Ben 25 μM and 25 nM/25 μM Ben X Ben50 μM and 500 nM/50 μM Ben X Ben 50 μM and 5 nM/50 μM Ben X Ben 50 μMand 25 nM/50 μM Ben X Ben 100 μM and 500 nM/100 μM Ben Ben 100 μM and 5nM/100 μM Ben Ben 100 μM and 25 nM/100 μM Ben Q-10 25 μM and 500 nM/25μM Q-10 X Q-10 25 μM and 5 nM/25 μM Q-10 X Q-10 25 μM and 25 nM/25 μMQ-10 X Q-10 50 μM and 500 nM/50 μM Q-10 X Q-10 50 μM and 5 nM/50 μM Q-10X Q-10 50 μM and 25 nM/50 μM Q-10 X Q-10 100 μM and 500 nM/100 μM Q-10 XQ-10 100 μM and 5 nM/100 μM Q-10 X Q-10 100 μM and 25 nM/100 μM Q-10 X

The data indicates the addition of phenylacetate in combination withbenzoquinone significantly decreases the cellular proliferation. Thecombination with CoQ10 and phenylacetate significantly decrease the cellnumber compared to incubation with CoQ10 and benzoquinone alone.

Cell Proliferation Assay of the Combination of 4-Hydroxy-Benzoate withFarnesyl: 4-Hydroxy-Benzoate was incubated in combination with Farnesyl.The summary of the results are listed below. 4-Hydroxybenzoate groupswere compared to the control and Farnesyl control groups. The Xsignifies a statistical decrease in cell number.

TABLE 58 4-Hydroxy-Benzoate and/or Farnesyl Compared to 4- Hydroxy toCompared to 4-Hydroxy- Compared compound w/o Farnesyl Benzoate to CtrlFarnesyl Control 500 nm X 500 nm w/ X Farnesyl (35 μM) 500 nm w/ XFarnesyl (70 μM)  1 μm Error  1 μm w/Farnesyl Error (35 μM)  1 μmw/Farnesyl Error (70 μM)  50 μm X 50 μm w/ Farnesyl X (35 μM) 50 μm w/Farnesyl X (70 μM)

Cell Proliferation Assay of the Combination of L-Phenylalanine withBenzoquinone: A cell proliferation assay was performed to test thecombination of L-Phenylalanine combined with Benzoquinone. Below is asummary of the results of L-Phenylalanine compared to the control andBenzoquinone control. The X signifies a statistical decrease.

TABLE 59 L-Phenylalanine and/or Benzoquinone Compared to L-Phenylalanine to Compared to Compared compound w/o BenzoquinoneL-Phenylalanine to Ctrl Benzoquinone Control  5 μM  5 μm w/Benzo X  (50μM)  5 μm w/Benzo X (100 μM)  25 μm  25 μm w/Benzo X  (50 μM)  25 μmw/Benzo X (100 μM) 100 μm 100 μm w/Benzo X X X  (50 μM) 100 μm w/Benzo XX X (100 μM)

A similar synergistic role is seen for the L-Phenylalanine combined withBenzoquinone.

Cell Proliferation Assay of the Combination of L-Phenylalanine withFarnesyl: Preliminary results for combination cell proliferation studyof L-Phenylalanine incubated in combination with Farnesyl. TheL-Phenylalanine were compared to the control and Farnesyl control group.An X signifies a statistical decrease in cell number.

TABLE 60 L-Phenylalanine and/or Farnesyl Compared to L- Phenylalanine toCompared to Compared compound w/o Farnesyl L-Phenylalanine to CtrlFarnesyl Control  5 μM  5 μm w/Farnesyl  (50 μM)  5 μm w/Farnesyl (100μM)  25 μm X 25 μm w/Farnesyl X X X  (50 μM) 25 μm w/Farnesyl X X X (100μM) 100 μm X 100 μm w/ X X Farnesyl (50 μM) 100 μm w/ X Farnesyl (100μM)

Cell Proliferation Assay of the Combination of L-Tyrosine withBenzoquinone: L-Tyrosine was incubated in combination with Benzoquinoneafter which a cell count was performed. The groups were compared thecontrol groups and Benzoquinone control group.

TABLE 61 L-Tyrosine and/or Benzoquinone Compared to L- Tyrosine toCompared to Compared compound w/o Benzoquinone L-Tyrosine to CtrlBenzoquinone Control 500 nm 500 nm w/Benzo  (50 μM) 500 nm w/Benzo (100μM)  5 μm X  5 μm w/Benzo X (50 μM)  5 μm w/Benzo X (100 μM)  15 μm X 15 μm w/Benzo X  (50 μM)  15 μm w/Benzo x (100 μM)

The addition of Benzoquinone did not amplify the effect of L-Tyrosine onthe cell number.

Cell Proliferation Assay of the Combination of L-Tyrosine withBenzoquinone: This study examined the combination of L-Tyrosine withFarnesyl. The groups were compared to control and Farnesyl controlgroups.

TABLE 62 L-Tyrosine and/or Farnesyl Compared to L- Tyrosine to Comparedto Compared compound w/o Farnesyl L-Tyrosine to Ctrl Farnesyl Control500 nm 500 nm w/Farnesyl  (50 μM) 500 nm w/Farnesyl  (50 μM)  5 μm X  5μm w/Farnesyl X  (50 μM)  5 μm w/Farnesyl X (100 μM)  15 μm X  15 μmw/Farnesyl X  (50 μM)  15 μm w/Farnesyl X (100 μM)

Combining L-Tyrosine and Farnesyl does not appear to have a synergisticeffect on reducing the cell number in this experiment.

The synthesis of the CoQ10 is divided into two main parts, which consistof the synthesis of the ring structure and synthesis of the side chainstructure. Here, oncogenic cells were supplemented with compounds whichare precursors for the synthesis of the side chain and the ringstructure components. These results have focused the study to 3 maincomponents involved in the synthesis of the ring structure and twocompounds that play a role in the attachment of the ring structure tothe side chain structure. The three compounds that have shown asignificant reduction in Bcl-2 and increase in Caspase-3 expressionare: 1) L-Phenylalanine, 2) L-Tyrosine and 3) 4-Hydroxyphenylpyruvate.The two compounds involved with the attachment of the side chain to thering structure are: 1) 4-hydroxy benzoate and 2) Phenylacetate.

These results also showed that exogenous delivery of these compounds incombination with 2,3 Dimethoxy-5-methyl-p-benzoquinone (benzoquinone)significantly inhibits cell proliferation. This indicates asupplementation of the ring structure with compounds for the attachmentof the side chain to the benzoquinone ring may supplement an impairedCoQ10 synthesis mechanism. This may also assist in the stabilization ofthe molecule to maintain the functional properties required by cellularprocesses. Phenylacetate is a precursor for the synthesis of4-Hydroxybenzoate, which exogenous delivery in combination withbenzoquinone has a similar effect in oncogenic cells.

Example 23 Modulation of Gene Expression by Coenzyme Q10 in Cell Modelfor Diabetes

Coenzyme Q10 is an endogenous molecule with an established role in themaintenance of normal mitochondrial function by directly influencingoxidative phosphorylation. Experimental evidence is presented thatdemonstrates the ability of Coenzyme Q10 in modulating intracellulartargets that serve as key indices of metabolic disorders, such asdiabetes, in a manner representative of therapeutic endpoints.

In order to understand how Coenzyme Q10 regulates expression of genesassociated with the cause or treatment of diabetes, immortalized primarykidney proximal tubular cell line derived from human kidney (HK-2) andprimary cultures of the human aortic smooth muscle cells (HASMC) wereused as experimental models. The HK-2 and HASMC cells are normallymaintained in culture at 5.5 mM glucose, which is a concentration thatcorresponds to a range considered normal in human blood. However, inorder to simulate a diabetic environment, both cell lines weresubsequently maintained at 22 mM glucose, which corresponds to the rangeobserved in human blood associated with chronic hyperglycemia. The cellswere subsequently allowed to propagate over 3 passages so that theintracellular regulation processes were functionally adapted to mimic adiabetic state. The choice of cell line was based on the physiologicinfluence of diabetes on renal dysfunction and progression to end-stagerenal disease (ESRD) in addition to the progressive pathophysiology of acompromised cardiovascular function.

Effect of Coenzyme Q10 on Gene Expression in HK-2 Cells using theDiabetes PCR Array

The Diabetes PCR array (SABiosciences) offers a screen for 84 genessimultaneously. The 4 treatments tested in this study were:

-   -   HK-2;    -   HK-2H maintained 22 mM glucose;    -   HK2(H)+50 μM Coenzyme Q10; and    -   HK2(H)+100 μM Coenzyme Q10.

A stringent analysis of the Real time PCR data of the HK-2 samples onthe Diabetes Arrays (Cat # PAHS-023E, SABiosciences Frederick Md.) wasmade to exclude all results where gene regulation was not at least atwo-fold regulation over HK-2 normal untreated cells with a p value ofless than 0.05. Genes that were observed to be regulated either bychronic hyperglycemia or by Coenzyme Q10 are listed in Table 63 andtheir functions and subcellular locations (derived from IngenuityPathway Analysis) are listed in Table 64.

TABLE 63 HK-2(H) HK-2(H)-50 μM HK-2(H)-100 μM Fold Coenzyme Q10 CoenzymeQ10 Genes regulation p value Fold regulation p value Fold regulation pvalue CEACAM1 1.26 0.409 3.47 0.067 5.36 0.032 PIK3C2B 1.48 0.131 2.320.115 3.31 0.003 INSR −1.09 0.568 2.51 0.103 2.88 0.024 TNF 2.00 0.0052.57 0.042 2.81 0.020 ENPP1 −1.50 0.002 1.42 0.238 2.67 0.038 PRKCB−1.75 0.005 1.82 0.280 2.49 0.042 DUSP4 1.27 0.318 1.24 0.455 2.26 0.060SELL −1.58 0.219 1.77 0.042 2.06 0.021 SNAP25 −1.00 0.934 1.46 0.3771.97 0.059

TABLE 64 Symbol Entrez Gene Name Location Type(s) CEACAM1carcinoembryonic antigen- Plasma trans- related cell adhesion mole-Membrane membrane cule 1 (biliary glycoprotein) receptor PIK3C2Bphosphoinositide-3-kinase, Cytoplasm kinase class 2, beta polypeptideINSR insulin receptor Plasma kinase Membrane TNF tumor necrosis factor(TNF Extracellular cytokine superfamily, member 2) Space ENPP1ectonucleotide pyrophos- Plasma enzyme phatase/phosphodiesterase 1Membrane PRKCB protein kinase C, beta Cytoplasm kinase DUSP4 dualspecificity phosphatase 4 Nucleus phosphatase SELL selectin L Plasmaother Membrane SNAP25 synaptosomal-associated Plasma transporterprotein, 25kDa Membrane

Among the detected RNA transcripts with modulated levels, the CarcinoEmbryonic Antigen Cell Adhesion Molecule 1 (CEACAM1) was identified asbeing highly upregulated in HK2(H) cells, particularly with 100 μMCoenzyme Q10 treatment. CEACAM-1, also known as CD66a and BGP-I, is a115-200 KD type I transmembrane glycoprotein that belongs to themembrane-bound CEA subfamily of the CEA superfamily. On the surface ofcells, it forms noncovalent homo- and heterodimers. The extracellularregion contains three C2-type Ig-like domains and one N-terminal V-typeIg-like domain. Multiple splice variants involving regions C-terminal tothe second C2-type domain (aa 320 and beyond) exist. The lack of intactCEACAM1 expression in mice has been proposed to promote the metabolicsyndrome associated with diabetes, while an increase in expression ofCEACAM1 is associated with increased insulin internalization, whichsuggests an increase in insulin sensitivity and glucose utilization(e.g., movement of glucose from blood into the cells), thus mitigatinginsulin resistance, a hallmark characteristic of type 2 diabetesmellitus.

As shown in Table 63, insulin receptor (INSR) expression was alsoaltered in diabetic HK-2 cells treated with Coenzyme Q10. Without beingbound by theory, the increase in expression of INSR with Coenzyme Q10treatment should enhance insulin sensitivity (either alone or inaddition to expression of CEACAM1) with the potential to reverse a majorphysiologic/metabolic complication associated with diabetes.

Effect of Coenzyme Q10 on Gene Expression in HK-2 Cells usingMitochondrial Arrays

Differential expression of mitochondrial genes in diabetes was assayedusing the mitochondria arrays (Cat# PAHS 087E, SABisociences FrederickMd.). Genes that were regulated by chronic hyperglycemia and/or CoenzymeQ10 treatment are listed in Table 65 while their functions and locationare included in Table 66.

TABLE 65 HK2 (H) HK-2(H) 50 μM HK-2(H) 100 μM Genes untreated p valueCoenzyme Q10 p value Coenzyme Q10 p value GRPEL1 −1.5837 0.151255−2.6512 0.04704 −1.933 0.139161 SLC25A3 −8.6338 0.071951 −8.2059 0.0425−1.6984 0.995194 TOMM40 −2.3134 0.140033 −1.1567 0.115407 −1.95090.038762 TSPO −3.6385 0.111056 −6.7583 0.073769 −2.1104 0.167084

TABLE 66 Symbol Entrez Gene Name Location Type(s) GRPEL1 GrpE-like 1,mito- Mitochondria other chondrial (E.coli) SLC25A3 solute carrierfamily 25 Mitochondrial transporter (mitochondrial carrier; membrane.phosphate carrier), member 3 TOMM40 translocase of outer Outer membraneion channel mitochondrial membrane of mitochondria. 40 homolog (yeast)TSPO translocator protein Outer membrane transmembrane (18kDa) ofmitochondria. receptor

To date, the role of the four mitochondrial genes identified (Table 65)in diabetic HK-2 cells treated with Coenzyme Q10 in diabetes isuncharacterized.

Study 2: Effect of Coenzyme Q10 on Gene Expression in HASMC Cells usingthe Diabetes PCR Array

The Diabetes PCR array (SABiosciences) offers a screen for 84 genessimultaneously. The 4 treatments tested in this study were:

-   -   HASMC;    -   HASMC H maintained at 22 mM glucose;    -   HASMC (H)+50 μM Coenzyme Q10; and    -   HASMC (H)+100 μM Coenzyme Q10.

A stringent analysis of the Real time PCR data of the HASMC cell sampleson the Diabetes Arrays (Cat #PAHS-023E, SABiosciences Frederick Md.) wasmade to exclude all results where gene regulation was not at least atwo-fold regulation over HASMC normal untreated cells with a p value ofless than 0.05. Genes that were observed to be regulated either bychronic hyperglycemia or by Coenzyme Q10 are listed in Table 67.

TABLE 67 HASMC-(H)-50 μM HASMC-(H)-100 μM Genes HASMC-(H) p valueCoenzyme Q10 p value Coenzyme Q10 p value AGT 1.3051 0.547507 −1.01690.781622 2.3027 0.030195 CCL5 −17.4179 0.013798 −5.3796 0.022489 −4.69130.022696 CEACAM1 −5.5629 0.012985 −5.3424 0.014436 −5.8025 0.012948 IL62.7085 0.049263 3.8172 0.012685 6.0349 0.000775 INSR 1.4649 0.2077881.9622 0.081204 2.0801 0.016316 NFKB1 1.482 0.072924 1.3779 0.1911912.0898 0.027694 PIK3C2B 2.0479 0.218276 1.4331 0.254894 2.6329 0.069422SELL −1.9308 0.087513 1.2476 0.393904 4.0371 0.000177 TNF −1.8140.108322 −3.2434 0.043526 −1.8489 0.133757

In HASMC cells, treatment of hyperglycemic cells with Coenzyme Q10resulted in the altered expression of genes involved in regulatingvascular function (AGT), insulin sensitivity (CEACAM1, INSR, SELL) andinflammation/immune function (IL-6, TNF, CCL5). Without being bound bytheory, an increase in expression of INSR may be associated withincreased insulin sensitivity in HASMC cells, which is a physiologicalproperty that would be beneficial in the treatment of diabetes, whileIL-6, in addition to its immunoregulatory properties, has been proposedto affect glucose homeostasis and metabolism, both directly andindirectly, by action on skeletal muscle cells, adipocytes, hepatocytes,pancreatic β-cells and neuroendocrine cells. Upon activation, normalT-cell express and secrete RANTES and chemokine(C-Cmotif) ligand (CCL5).CCL5 is expressed by adipocytes, and serum levels of RANTES areincreased in obesity and type 2 diabetes. However, as shown in Table 67,treatment of HASMC cells with Coenzyme Q10 causes a significant decreasein the expression of CCL5. Based on the foregoing data, it is expectedthat administration of Coenzyme Q10 will have a therapeutic benefit inthe management of diabetes.

Effect of Coenzyme Q10 on Gene Expression in HASMC Cells UsingMitochondrial Arrays

Differential expression of mitochondrial genes in diabetes was assayedusing the mitochondria arrays (Cat# PAHS 087E, SABisociences FrederickMd.). Genes that were regulated by chronic hyperglycemia and/or CoenzymeQ10 treatment are shown in Table 68.

TABLE 68 HASMC-(H) 50 μM HASMC-(H)-100 μM Genes HASMC-(H) p valueCoenzyme Q10 p value Coenzyme Q10 p value BCL2L1 −1.6558 0.244494−2.7863 0.008744 −2.3001 0.014537 MFN1 −1.4992 0.317009 −1.2585 0.021185−2.2632 0.005961 PMAIP1 −4.7816 0.206848 −6.8132 0.000158 −4.3520.000286 SLC25A1 −2.2051 0.020868 −1.834 0.00581 −3.0001 0.03285SLC25A13 −2.0527 0.035987 −1.5 0.029019 −1.5245 0.043712 SLC25A19−1.0699 0.417217 −1.4257 0.104814 −2.1214 0.007737 SLC25A22 −2.17470.007344 −1.9839 0.0013 −10.3747 0.003437 TIMM44 −1.3605 0.414909−2.3214 0.004118 −1.9931 0.010206 TOMM40 −1.1982 0.428061 −2.09220.002195 −2.2684 0.003272 TSPO −1.402 0.304875 −2.0586 0.061365 −2.36470.044656

Treatment of hyperglycemic HASMC cells with Coenzyme Q10 resulted inaltered expression of genes that regulate programmed cell death orapoptosis (BCL2L1, PMIAP1 also known as NOXA), transporter proteins(SLC25A1 [citrate transporter], SLC25A13 [aspartate-glutamateexchanger], SLC25A19 [thiamine pyrophosphate transporter] and SLC25A22[glutamate-hydrogen cotransporter]) and mitochondrial matrix transportproteins (MFN1, TIMM44 and TOMM40). The activities of these transportersplay important role in the regulation of precursors essential for theKreb's cycle and maintenance of mitochondrial oxidative phosphorylation.These results indicate that exposure of diabetic HASMC cells to CoenzymeQ10 is associated with changes in expression of cytoplasmic andmitochondrial genes, which in turn is consistent with Coenzyme Q10providing a therapeutic benefit in the treatment of diabetes.

A comparison of the data obtained by treating HASMC cells and HK-2 cellswith Coenzyme Q10 or in a hyperglycemic environment reveals that 4 geneswere commonly regulated by Coenzyme Q10 in both cell lines (e.g.,PIK3C2B and SELL in the gene expression assay and TOMM40 and TSPO in themitochondrial array assay). These results demonstrate that treatment ofcells with Coenzyme Q10 in a diabetic environment is associated withaltered expression of genes that are known to be involved in the causeor treatment of diabetes.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the following claims.

1. A method for treating, alleviating symptoms of, inhibitingprogression of, or preventing a metabolic disorder in a mammal, themethod comprising: administering to the mammal in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising at least one environmental influencer (env-influencer),wherein the environmental influencer selectively elicits, in a diseasecell of the mammal, a cellular metabolic energy shift towards normalizedmitochondrial oxidative phosphorylation.
 2. The method of claim 1,wherein the environmental influencer does not substantially elicit, innormal cells of the mammal, the cellular metabolic energy shift towardsmitochondrial oxidative phosphorylation.
 3. The method of claim 1,wherein the mammal is human (or a non-human mammal).
 4. The method ofclaim 1, wherein the metabolic disorder is responsive or sensitive totreatment by Coenzyme Q10 or its metabolites or analogs thereof.
 5. Themethod of claim 1, wherein the metabolic disorder is characterized by adysregulated mitochondrial oxidative phosphorylation function that leadsto altered gene regulation and/or protein-protein interactions whichcontribute to or causally lead to the metabolic disease.
 6. The methodof claim 1, wherein the environmental influencer comprises: (a)benzoquinone or at least one molecule that facilitates the biosynthesisof the benzoquinone ring, and (b) at least one molecule that facilitatesthe synthesis of and/or attachment of isoprenoid units to thebenzoquinone ring.
 7. The method of claim 6, wherein said at least onemolecule that facilitates the biosynthesis of the benzoquinone ringcomprises: L-Phenylalanine, DL-Phenylalanine, D-Phenylalanine,L-Tyrosine, DL-Tyrosine, D-Tyrosine, 4-hydroxy-phenylpyruvate,3-methoxy-4-hydroxymandelate (vanillylmandelate or VMA), vanillic acid,pyridoxine, or panthenol.
 8. The method of claim 6, wherein said atleast one molecule that facilitates the synthesis of and/or attachmentof isoprenoid units to the benzoquinone ring comprises: phenylacetate,4-hydroxy-benzoate, mevalonic acid, acetylglycine, acetyl-CoA, orfarnesyl.
 9. The method of claim 1, wherein the environmental influencercomprises: (a) one or more of L-Phenylalanine, L-Tyrosine, and4-hydroxyphenylpyruvate; and, (b) one or more of 4-hydroxy benzoate,phenylacetate, and benzoquinone.
 10. The method of claim 1, wherein theenvironmental influencer: (a) inhibits Bcl-2 expression and/or promotesCaspase-3 expression; and/or, (b) inhibits cell proliferation.
 11. Themethod of claim 1, wherein the environmental influencer is amultidimensional intracellular molecule (MIM).
 12. The method of claim11, wherein the MIM is selected from: alpha ketoglutarate/alphaketoglutaric acid, Malate/Malic acid, Succinate/Succinic acid,Glucosamine, Adenosine, Adenosine Diphosphate, Glucuronide/Glucuronicacid, Nicotinic Acid, Nicotinic Acid Dinucleotide,Alanine/Phenylalanine, Pyridoxine, Thiamine, or Flavin AdenineDinucleotide.
 13. The method of claim 1, wherein the environmentalinfluencer is an epimetabolic shifter (epi-shifter).
 14. The method ofclaim 13, wherein the epimetabolic shifter is selected from:Transaldolase, Transketolase, Succinyl CoA synthase, PyruvateCarboxylase, or Riboflavin.
 15. The method of claim 13, wherein theepimetabolic shifter is coenzyme Q10.
 16. The method of claim 1, whereinthe concentration of the environmental influencer in the tissues of thehuman being treated is different than that of a control standard ofhuman tissue representative of a healthy or normal state.
 17. The methodof claim 1, wherein the form of the environmental influenceradministered to the human is different than the predominant form foundin systemic circulation in the human.
 18. The method of claim 1, whereinthe amount sufficient to treat the metabolic disorder in the humanup-regulates or down-regulates mitochondrial oxidative phosphorylation.19. The method of claim 18, wherein the amount sufficient to treat themetabolic disorder in the human modulates anaerobic use of glucoseand/or lactate biosynthesis.
 20. The method of claim 1, wherein thetreatment occurs via an interaction of the env-influencer withHNF4alpha.
 21. The method of claim 1, wherein the treatment occurs viaan interaction of the env-influencer with transaldolase.
 22. The methodof claim 1, wherein the metabolic disorder is selected from the groupconsisting of diabetes, obesity, pre-diabetes, Metabolic Syndrome andany key elements of a metabolic disorder.
 23. The method of claim 22,wherein the metabolic disorder is diabetes, and the env-influenceraffects beta cell function, insulin metabolism, and/or glucagondeposition.
 24. The method of claim 22, wherein the metabolic disorderis obesity, and the env-influencer affects beta cell oxidation in themitochondria, decrease in adipocyte size, and/or control of cortisollevels.
 25. The method of claim 22, wherein the metabolic disorder is acardiovascular disease, and the env-influencer affects decrease insmooth muscle cell proliferation in the tunica media, lipidperoxidation, thromboxane-ax2 synthesis, TNFα, IL-1B, plateletaggregation, decrease in nitric oxide (NO) production, plaque depositionand/or normalized glycemic control.
 26. The method of claim 22, whereinsaid key elements of a metabolic disorder is selected from the groupconsisting of impaired fasting glucose, impaired glucose tolerance,increased waist circumference, increased visceral fat content, increasedfasting plasma glucose, increased fasting plasma triglycerides,decreased fasting high density lipoprotein level, increased bloodpressure, insulin resistance, hyperinsulinemia, cardiovascular disease,arteriosclerosis, coronary artery disease, peripheral vascular disease,cerebrovascular disease, congestive heart failure, elevated plasmanorepinephrine, elevated cardiovascular-related inflammatory factors,elevated plasma factors potentiating vascular endothelial dysfunction,hyperlipoproteinemia, arteriosclerosis or atherosclerosis, hyperphagia,hyperglycemia, hyperlipidemia, and hypertension or high blood pressure,increased plasma postprandial triglyceride or free fatty acid levels,increased cellular oxidative stress or plasma indicators thereof,increased circulating hypercoagulative state, hepatic steatosis,hetaptic steatosis, renal disease including renal failure and renalinsufficiency.
 27. The method of claim 1, further comprisingadministering an additional therapeutic agent.
 28. The method of claim27, wherein the additional therapeutic agent is selected from the groupconsisting of diabetes mellitus-treating agents, diabetic complicationtreating agents, anti-hyperlipemic agents, hypotensive orantihypertensive agents, anti-obesity agents, diuretics,chemotherapeutic agents, immunotherapeutic agents and immunosuppressiveagents.
 29. A method for selectively augmenting mitochondrial oxidativephosphorylation, in a disease cell of a mammal in need of treatment fora metabolic disorder, the method comprising: administering to saidmammal a therapeutically effective amount of a pharmaceuticalcomposition comprising at least one env-influencer, thereby selectivelyaugmenting mitochondrial oxidative phosphorylation in said disease cellof the mammal.
 30. The method of claim 29, further comprisingup-regulating the expression of one or more genes selected from thegroup consisting of the molecules listed in Tables 2-4 & 6-28 & 63-68having a positive fold change; and/or down-regulating the expression ofone or more genes selected from the group consisting of the moleculeslisted in Tables 2-4 and 6-28 & 63-68 having a negative fold change. 31.The method of claim 29, further comprising modulating the expression ofone or more genes selected from the group consisting of HNF4-alpha,Bcl-xl, Bcl-xS, BNIP-2, Bcl-2, Birc6, Bcl-2-L11, XIAP, 20 BRAF, Bax,c-Jun, Bmf, PUMA, cMyc, transaldolase 1, COQ1, COQ3, COQ6,prenyltransferase, 4-hydrobenzoate, neutrophil cytosolic factor 2,nitric oxide synthase 2A, superoxide dismutase 2, VDAC, Bax channel,ANT, Cytochrome c, complex 1, complex II, complex III, complex IV, Foxo3a, DJ-1, IDH-1, Cpt1C and Cam Kinase II.
 32. The method of anyone ofclaims 29-31, wherein the metabolic disorder is selected from the groupconsisting of diabetes, obesity, pre-diabetes, Metabolic Syndrome andany key elements of a metabolic disorder.
 33. The method of anyone ofclaims 29-31, wherein said key elements of a metabolic disorder isselected from the group consisting of impaired fasting glucose, impairedglucose tolerance, increased waist circumference, increased visceral fatcontent, increased fasting plasma glucose, increased fasting plasmatriglycerides, decreased fasting high density lipoprotein level,increased blood pressure, insulin resistance, hyperinsulinemia,cardiovascular disease, arteriosclerosis, coronary artery disease,peripheral vascular disease, cerebrovascular disease, congestive heartfailure, elevated plasma norepinephrine, elevated cardiovascular-relatedinflammatory factors, elevated plasma factors potentiating vascularendothelial dysfunction, hyperlipoproteinemia, arteriosclerosis oratherosclerosis, hyperphagia, hyperglycemia, hyperlipidemia, andhypertension or high blood pressure, increased plasma postprandialtriglyceride or free fatty acid levels, increased cellular oxidativestress or plasma indicators thereof, increased circulatinghypercoagulative state, hepatic steatosis, hetaptic steatosis, renaldisease including renal failure and renal insufficiency.
 34. The methodof claim 29, further comprising administering an additional therapeuticagent.
 35. The method of claim 34, wherein the additional therapeuticagent is selected from the group consisting of diabetesmellitus-treating agents, diabetic complication treating agents,antihyperlipemic agents, hypotensive or antihypertensive agents,antiobesity agents, diuretics, chemotherapeutic agents,immunotherapeutic agents and immunosuppressive agents.
 36. A method ofidentifying an agent that is effective in treating a metabolic disorder,the method comprising: (1) selecting an environmental influencer; (2)identifying an environmental influencer capable of shifting themetabolic state of a cell; and (3) determining whether the environmentalinfluencer is effective in treating the metabolic disorder; therebyidentifying an agent that is effective in treating a metabolic disorder.37. The method of claim 36, wherein an environmental influencer isidentified as capable of shifting the metabolic state of a cell bymeasuring changes in any one or more of mRNA expression, proteinexpression, lipid or metabolite concentration, levels of bioenergeticmolecules, cellular energetics, mitochondrial function and mitochondrialnumber.
 38. The method of claim 36, wherein an environmental influencereffective in treating a metabolic disorder is capable of reducingglucose levels or lipid levels in a patient.
 39. A compositioncomprising an agent identified according to the method of any one ofclaims 36-38.
 40. A kit comprising the composition of claim
 39. 41. Amethod of reducing glucose levels in a patient comprising administeringto the patient an effective amount of the composition of claim
 39. 42. Amethod of reducing lipid levels in a patient comprising administering tothe patient an effective amount of the composition of claim
 39. 43. Amethod for treating, alleviating symptoms of, inhibiting progression of,or preventing a Coenzyme Q10 responsive disorder in a mammal, the methodcomprising: administering to the mammal in need thereof atherapeutically effective amount of pharmaceutical compositioncomprising at least one environmental influencer (env-influencer),wherein the environmental influencer selectively elicits, in a diseasecell of the mammal, a cellular metabolic energy shift towards levels ofglycolysis and mitochondrial oxidative phosphorylation observed in anormal cell of the mammal under normal physiological conditions.
 44. Themethod of claim 43, wherein the Coenzyme Q10 responsive disorder is ametabolic disorder.